Biological targeting compositions and methods of using the same

ABSTRACT

Modified red blood cells are described. In an embodiment, the modified red blood cell includes a target-binding agent. Targeted delivery of imaging agents, drugs, and peptide and protein pharmaceuticals using modified red blood cells are described. Processes for preparing the modified red blood cells, pharmaceutical and diagnostic compositions containing the same and methods of diagnosis and treatment involving the modified red blood cells are described.

CROSS-REFERENCE TO RELATED APPLICATIONS Related Applications

The present application constitutes is related to U.S. patentapplication Ser. No. To be Assigned, titled BIOLOGICAL TARGETINGCOMPOSITIONS AND METHODS OF USING THE SAME, naming Roderick A. Hyde,Muriel Y. Ishikawa, Edward K. Y. Jung, William Gates, Alois A. Langer,Eric C. Leuthardt, Royce A. Levien, Clarence T. Tegreene, Thomas A.Weaver, Charles Whitmer, Lowell L. Wood, Jr., and Victoria Y. H. Wood asinventors, filed 13 Aug. 2008, which is currently co-pending.

The present application constitutes is related to U.S. patentapplication Ser. No. To be Assigned, titled BIOLOGICAL TARGETINGCOMPOSITIONS AND METHODS OF USING THE SAME, naming Roderick A. Hyde,Muriel Y. Ishikawa, Edward K. Y. Jung, William Gates, Alois A. Langer,Eric C. Leuthardt, Royce A. Levien, Clarence T. Tegreene, Thomas A.Weaver, Charles Whitmer, Lowell L. Wood, Jr., and Victoria Y. H. Wood asinventors, filed 13 Aug. 2008, which is currently co-pending.

The present application constitutes is related to U.S. patentapplication Ser. No. To be Assigned, titled BIOLOGICAL TARGETINGCOMPOSITIONS AND METHODS OF USING THE SAME, naming Roderick A. Hyde,Muriel Y. Ishikawa, Edward K. Y. Jung, William Gates, Alois A. Langer,Eric C. Leuthardt, Royce A. Levien, Clarence T. Tegreene, Thomas A.Weaver, Charles Whitmer, Lowell L. Wood, Jr., and Victoria Y. H. Wood asinventors, filed 13 Aug. 2008, which is currently co-pending.

The present application constitutes is related to U.S. patentapplication Ser. No. To be Assigned, titled BIOLOGICAL TARGETINGCOMPOSITIONS AND METHODS OF USING THE SAME, naming Roderick A. Hyde,Muriel Y. Ishikawa, Edward K. Y. Jung, William Gates, Alois A. Langer,Eric C. Leuthardt, Royce A. Levien, Clarence T. Tegreene, Thomas A.Weaver, Charles Whitmer, Lowell L. Wood, Jr., and Victoria Y. H. Wood asinventors, filed 13 Aug. 2008, which is currently co-pending.

All subject matter of the Related Applications and of any and allparent, grandparent, great-grandparent, etc. applications of the RelatedApplications is incorporated herein by reference to the extent suchsubject matter is not inconsistent herewith.

SUMMARY

In one aspect, a modified red blood cell is described, which modifiedred blood cell comprises a red blood cell, one or more fusion molecules,and at least one target-binding agent comprising a target recognitionmoiety, wherein the target recognition moiety is designed to recognizeone or more target cells, and wherein the one or more fusion moleculesare designed to participate in fusion of the red blood cell with thetarget cell.

In an embodiment, the modified red blood cell is genetically engineeredto express one or more of the at least one target-binding agent and theone or more fusion molecules. In an embodiment, the at least onetarget-binding agent and the one or more fusion molecules is associatedwith the cell surface of the red blood cell.

In an embodiment, the one or more fusion molecules includes, but is notlimited to, at least one of: an antigen; ligand; receptor; polyamide;peptide; carbohydrate; oligosaccharide; polysaccharide; low densitylipoprotein (LDL) or an apoprotein of LDL; steroid; steroid derivative;hormone; hormone-mimic; lectin; drug; antibiotic; aptamer; DNA; RNA;lipid; an antibody; and an antibody-related polypeptide. In anembodiment, the one or more fusion molecules is a syncytin-1 protein.

In an embodiment, the target recognition moiety includes, but is notlimited to, at least one of: an antigen; ligand; receptor; polyamide;peptide; carbohydrate; oligosaccharide; polysaccharide; low densitylipoprotein (LDL) or an apoprotein of LDL; steroid; steroid derivative;hormone; hormone-mimic; lectin; drug; antibiotic; aptamer; DNA; RNA;lipid; an antibody; and an antibody-related polypeptide.

In an embodiment, the modified red blood cell may include one or moreactivatable molecular markers associated with the red blood cell,wherein the one or more molecular markers is activated by theinteraction of the red blood cell with the one or more target cells, andwherein the activated molecular markers are capable of producing adetectable response. For example, the one or more molecular markers is aphotoactivatable molecule and the detectable response is the productionof reactive singlet oxygen molecules. In an embodiment, the red bloodcell may be genetically engineered to express the one or more molecularmarkers, e.g., an aptamer based molecular beacon.

In an embodiment, the at least one target-binding agent may include aphotoactivatable molecule and a quencher molecule coupled to the targetrecognition moiety, wherein the target-binding agent emits at least onesinglet oxygen radical molecule upon exposure to light of a suitablewavelength when the target-binding agent is bound to a target cell. Inan embodiment, the photoactivatable molecule and the quenching moleculeincludes a linking component. In an embodiment, the linking componentincludes an oligonucleotide of about 20-60 residues or a linker to linkwith an amino or hydroxy fatty acid or a sulfonic acid of from 1 to 20carbon atoms using an ester, an amide, or a sulfonamide linkage.

In an embodiment, the photoactivatable molecule includes, but is notlimited to, at least one of a: porphyrin; chlorin; bacteriochlorin;isbacteriochlorin; phthalocyanine; napthalocyanine; porphycene;porphycyanine; tetra-macrocyclic compound; poly-macrocyclic compound;pyropheo-phorbide; pentaphyrin; sapphyrin; texaphyrin; metal complexe;tetrahydrochlorin; phonoxazine dye; phenothiazine; chaloorganapyryliumdye; rhodamine; fluorescene; azoporphyrin; benzochlorin; purpurin;chlorophyll; verdin; triarylmethane; angelicin; chalcogenapyrillium dye;chlorophyll; coumarin; cyanine; ceratin daunomycin; daunomycinone;5-iminodauno-mycin; doxycycline; furosemide; gilvocarcin M; gilvocarcinV; hydroxy-chloroquine sulfate; lumidoxycycline; mefloquinehydrochloride; mequitazine; merbromin (mercurochrome); primaquinediphosphate; quinacrine dihydrochloride; quinine sulfate; tetracyclinehydrochloride; flavins; alloxazine; flavin mononucleotide;3-hydroxyflavone; limichrome; limiflavin; 6-methylalloxazine;7-methylalloxazine; 8-methylalloxazine; 9-methylalloxazine; 1-methyllimichrome; methyl-2-methoxybenzoate; 5-nitrosalicyclic acid;proflavine; and riboflavin; metallo-porphyrin; metallophthalocyanine;methylene blue derivative; naphthalmide; naphthalocyanine; pheophorbide;pheophytin; photosensitizer dimer and conjugate; phthalocyanine;porphycene; quinone; retinoid; rhodamine; thiophene; verdin; vitamin;and xanthene dye.

In an embodiment, the quencher molecule includes, but is not limited to,at least one of: 4-(4′-dimethylamino-phenylazo)benzoic acid (DABCYL);dabcyl succinimidyl ester; 4-(4′-dimethylamino-phenylazo)sulfonic(DABSYL); dabsyl succinimidyl ester; tetramethyl-rhodamaine (TAMRA);4-[(4-nitrophenyl)diazinyl]-phenylamine and4-[4-nitrophenyl)diazinyl]-naphthylamine; dabcylnitro-thiazole;6-(N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl]amino) hexanoic acid;6-carboxy-X-rhodamine (ROX);QSY-7;2-[4-(4-nitrophenylazo)-N-ethylphenyl-amino]ethanol (Disperse Red 1);2-[4-(2-chloro-4-nitrophenyl-azo)-N-ethylphenylamino]-ethanol (DisperseRed 13); tetrarhodamine isothiocyanate (TRITC); allophycocyanin;β-carotene; diarylrhodamine derivatives, QSY 7, QSY 9, QSY 21 dyes; QSY35 acetic acid succinimidyl ester; QSY 35 iodoacetamide; aliphaticmethylamine; napthalate; Reactive Red 4; and Malachite Green.

In an embodiment, the photoactivatable molecule may be linked to thequenching molecule and the target recognition moiety and configured sothat the photoactivatable molecule is quenched until the targetrecognition moiety is bound to the target molecule, whereupon thequenching molecule moves away from the photoactivatable molecule,enabling detectable excitation of the photoactivatable molecule uponirradiation with the light of a suitable wavelength.

In an embodiment, the red blood cell includes, but is not limited to, atleast one of: a reticulocyte; a red blood cell; and a fusion red bloodcell including between a red blood cell autologous to a subject and oneor more allogeneic erythrocytes, liposomes or artificial vesicles.

In an embodiment, the red blood cell may be genetically engineered toexpress one or more protein-based pharmaceuticals or one or moreRNA-based pharmaceuticals. In an embodiment, the red blood cell may beloaded with one or more pharmaceutical or imaging molecules. In anembodiment, the pharmaceutical molecule includes, but is not limited to,at least one of: an antibiotic, an antiviral agent, an antifungal agent,an antimicrobial, a polypeptide, an anti-parasitic agent, an antibody,an antibody-related polypeptide, an antineoplastic agent; aprotein-based agent; and a nucleic acid-based agent. In an embodiment,the red blood cell may be loaded with biologically-active entities, suchas for example, viruses and/or bacterium. Loaded viruses may be designedto infect a certain cellular target, and may be designed as vehicle forefficient gene delivery to a cellular target.

In an embodiment, the antineoplastic agent includes, but is not limitedto, at least one of: an alkylating agent; cisplatin; carboplatin;oxaliplatin; mechlorethamine; cyclophosphamide; chlorambucil;anti-metabolite compound; azathioprine; mercaptopurine; alkaloid;terpenoid; vinca alkaloid; vincristine; vinblastine; vinorelbine;vindesine; podophyllotoxin; taxanes; docetaxel; paclitaxel;topoisomerase inhibitors; camptothecin; irinotecan; topotecan;amsacrine; etoposide; etoposide phosphate; teniposide;epipodophyllotoxins; antitumour antibiotics; dactinomycin; trastuzumab,cetuximab, rituximab; bevacizumab; finasteride; tamoxifen;gonadotropin-releasing hormone agonists (GnRH); and goserelin.

In an embodiment, the antibiotic includes, but is not limited to, atleast one of: aminoglycoside; amikacin; gentamicin; kanamycin; neomycin;netilmicin; steptomycin; tobramycin; ansamycins; geldanamycin;herbimycin; carbacephem; loracarbef; carbacepenem; ertapenem; doripenem;imipenem/cilastatin; meropenem; cephalosporin; cefadroxil; cefazolin;cefalotin or cefalothin; cefalexin; cefaclor; cefamandole; cefoxitin;cefprozil; cefuroxime; cefixime; cefdinir; cefditoren; cefoperazone;cefotaxime; cefpodoxime; ceftazidime; ceftibuten; ceftizoxime;ceftriaxone; cefepime; ceftobiprole; glycopeptide; teicoplanin;vancomycin; macrolides; azithromycin; clarithromycin; dirithromycin;erythromicin; roxithromycin; troleandomycin; telithromycin;spectinomycin; monobactam; aztreonam; penicillins; amoxicillin;ampicillin; azlocillin; carbenicillin; cloxacillin; dicloxacillin;flucloxacillin; mezlocillin; meticillin; nafcillin; oxacillin;penicillin, piperacillin, ticarcillin; bacitracin; colistin; polymyxinB; quinolone; ciprofloxacin; enoxacin; gatifloxacin; levofloxacin;lomefloxacin; moxifloxacin; norfloxacin; ofloxacin; trovafloxacin;sulfonamide; mafenide; prontosil (archaic); sulfacetamide;sulfamethizole; sufanilimide (archaic); sulfasalazine; sulfisoxazole;trimethoprim; trimethoprim-sulfamethoxazole (co-trimoxazole) (TMP-SMX);tetracycline; demeclocycline; doxycycline; minocycline; oxytetracycline;tetracycline; arsphenamine; chloramphenicol; clindamycin; lincomycin;ethambutol; fosfomycin; fusidic acid; furazolidone; isoniazid;linezolid; metronidazole; mupirocin; nitrofuantoin; platensimycin;purazinamide; quinupristin/dalfopristin; rifampin or rifampicin; andtinidazole.

In an embodiment, the antiviral agent includes, but is not limited to,at least one of a: thiosemicarbazone; metisazone; nucleoside and/ornucleotide; acyclovir; idoxuridine; vidarabine; ribavirin; ganciclovir;famciclovir; valaciclovir; cidofovir; penciclovir; valganciclovir;brivudine; ribavirin, cyclic amines; rimantadine; tromantadine;phosphonic acid derivative; foscamet; fosfonet; protease inhibitor;saquinavir; indinavir; ritonavir; nelfinavir; amprenavir; lopinavir;fosamprenavir; atazanavir; tipranavir; nucleoside and nucleotide reversetranscriptase inhibitor; zidovudine; didanosine; zalcitabine; stavudine;lamivudine; abacavir; tenofovir disoproxil; adefovir dipivoxil;emtricitabine; entecavir; non-nucleoside reverse transcriptaseinhibitor; nevirapine; delavirdine; efavirenz; neuraminidase inhibitor;zanamivir; oseltamivir; moroxydine; inosine pranobex; pleconaril; andenfuvirtide.

In an embodiment, the anti-fungal agent includes, but is not limited to,at least one of a: allylamine; terbinafine; antimetabolite; flucytosine;azole; fluconazole; itraconazole; ketoconazole; ravuconazole;posaconazole; voriconazole; glucan synthesis inhibitor; caspofungin;micafungin; anidulafungin; polyenes; amphotericin B; amphotericin BLipid Complex (ABLC); amphotericin B Colloidal Dispersion (ABCD);liposomal amphotericin B (L-AMB); liposomal nystatin; and griseofulvin.

In an embodiment, the anti-parasitic agent includes, but is not limitedto, at least one of a: antiprotozoal agent; eflomithine; furazolidone;melarsoprol; metronidazole; ornidazole; paromomycin sulfate;pentamidine; pyrimethamine; tinidazole; antimalarial agent; quinine;chloroquine; amodiaquine; pyrimethamine; sulphadoxine; proguanil;mefloquine; halofantrine; primaquine; artemesinin and derivativesthereof; doxycycline; clindamycin; benznidazole; nifurtimox;antihelminthic; albendazole; diethylcarbamazine; mebendazole;niclosamide; ivermectin; suramin; thiabendazole; pyrantel pamoate;levamisole; piperazine family; praziquantel; triclabendazole;octadepsipeptide; and emodepside.

In an aspect, a pharmaceutical composition is described, thepharmaceutical composition comprising a modified red blood cell and apharmaceutically acceptable excipient.

In an aspect, a method of treatment is described, the method comprisingproviding a modified red blood cell to a subject, the modified red bloodcell comprising a red blood cell, one or more fusion molecules, and atleast one target-binding agent comprising a target recognition moiety,wherein the target recognition moiety is configured to recognize one ormore target cells including a neoplastic cell or a pathogen cell, andwherein the one or more fusion molecules are configured to participatein fusion of the red blood cell with the target cell.

In an embodiment, the modified red blood cell further comprises one ormore activatable molecular markers associated with the red blood cell,wherein the one or more molecular markers are configured to be activatedby the interaction of the red blood cell with the one or more targetcells, and are configured to provide a detectable response uponexcitation with electromagnetic energy.

In an embodiment, the method further comprises providing electromagneticenergy to the subject, the electromagnetic energy configured to induce adetectable response from the one or more activated molecular markersassociated with the red blood cell; and detecting the response from theone or more activated molecular markers.

In an embodiment, the at least one target-binding agent furthercomprises a photoactivatable molecule and a quencher molecule coupled tothe target recognition moiety, wherein the target-binding agent emits atleast one singlet oxygen radical molecule upon exposure to light of asuitable wavelength when the target-binding agent is bound to a targetcell.

In an embodiment, the method further comprises providing electromagneticenergy to a subject, the electromagnetic energy configured to activatethe photoactivatable molecules, wherein activation of thephotoactivatable molecule damages the target cells.

In an embodiment, activation of the one or more photoactivatablemolecules directly damages the one or more target cells. In anembodiment, activation of the one or more photoactivatable moleculesindirectly damages the one or more target cells.

In an embodiment, the neoplastic cell is associated with, but is notlimited to, at least one of: ovarian cancer; bladder cancer; lungcancer; cervical cancer; breast cancer; prostate cancer; glioma;fibrosarcoma; retinoblastoma; melanoma; soft tissue sarcoma;ostersarcoma; leukemia; colon cancer; carcinoma of the kidney;gastrointestinal cancer; salivary gland cancer and pancreatic cancer.

In an embodiment, the pathogen cell includes, but is not limited to, atleast one of: a bacteria; a fungi; a virus; and a parasite. In anembodiment, the bacteria includes, but is not limited to, at least oneof: Neisseria meningitides, Neisseria gonorrheoeae; Legionella; Vibriocholerae; Streptococci; Staphylococcus aureus; Staphylococcusepidermidis; Pseudomonas aeruginosa; Corynobacteria diphtheriae,Clostridium spp.; Eschericia coli; Bacillus anthracis; Bartonellahenselae; Bartonella quintana; Coxiella burneii; Chlamydia;Mycobacterium leprae; Salmonella; Shigella; Yersinia enterocolitica;Yersinia pseudotuberculosis; Legionella pneumophila; Mycobacteriumtuberculosis; Listeria monocytogenes; Mycoplasma spp.; Pseudomonasfluorescens; Vibrio cholerae; Haemophilus influenzae; Bacillusanthracis; Treponema pallidum; Leptospira; Borrelia; Corynebacteriumdiphtheriae; Francisell; Brucella melitensis; Campylobacter jejuni;Enterobacter; Proteus mirabilis; Proteus; and Klebsiella pneumoniae.

In an embodiment, the fungi includes, but is not limited to, at leastone of: Candida albicans; Candida glabrata; Aspergilus spp.; Torulopsisglabrata; Candida tropicalis; C. krusei; and C. parapsilosis.

In an embodiment, the virus includes, but is not limited to, at leastone of: adenovirus; coxsackievirus; hepatitis a virus; poliovirus;epstein-barr virus; herpes simplex; type 1; herpes simplex; type 2;human cytomegalovirus; human herpesvirus; type 8; varicella-zostervirus; hepatitis B virus; hepatitis C viruses; human immunodeficiencyvirus (HIV); influenza virus; measles virus; mumps virus; parainfluenzavirus; respiratory syncytial virus; papillomavirus; rabies virus; andRubella virus.

In an embodiment, the parasite includes, but is not limited to, at leastone of a: trypanosome; haemoprotozoa and parasites capable of causingmalaria; enteric and systemic cestode; taeniid cestod; entericcoccidian; enteric flagellate protozoa; filarial nematode;gastrointestinal and systemic nematode and hookworm.

In an aspect, a method for detecting a target in a biological sample isdescribed, the method comprising: (i) adding to the biological sample amodified red blood cell that binds to the target; and (ii) detecting themodified red blood cell wherein the modified red blood cell includes ared blood cell, one or more fusion molecules, and at least onetarget-binding agent comprising a target recognition moiety, wherein thetarget recognition moiety is designed to recognize one or more targetcells, and wherein the one or more fusion molecules are designed toparticipate in fusion of the red blood cell with the target cell.

In an aspect, a method of making a modified red blood cell is described,the method comprising: providing a red blood cell; contacting the redblood cell with one or more fusion molecules and one or moretarget-binding agents comprising a target recognition moiety. Further,the modified red blood cell may be isolated from the one or moretarget-binding agent and the one or more fusion molecules which are notassociated with the modified red blood cell, thereby making an isolatedmodified red blood cell.

In an embodiment, providing the red blood cell comprises differentiatingerythrocytes ex vivo from a stem cell or a reticulocyte.

In an aspect, a modified red blood cell comprising a red blood cell andat least one target-binding agent comprising a target recognition moietymay be coupled to a photoactivatable molecule and a quencher molecule,wherein the target-binding agent emits at least one singlet oxygenradical molecule upon exposure to light of a suitable wavelength whenthe target-binding agent is bound to a target molecule.

In an embodiment, the at least one target-binding agent is configured tobe associated with the cell surface of the red blood cell. In anembodiment, the at least one target-binding agent is configured to beinternalized within the red blood cell.

In an embodiment, the photoactivatable molecule and the quenchingmolecule include a linking component. In an embodiment, the linkingcomponent is an oligonucleotide of about 20-60 residues or is a linkerto link with an amino or hydroxy fatty acid or a sulfonic acid of fromabout 1 to 20 carbon atoms using an ester, amide, or sulfonamidelinkage.

In an embodiment, the at least one target recognition moiety includes,but is not limited to, at least one of: an antigen; ligand; receptor;polyamide; peptide; carbohydrate; oligosaccharide; polysaccharide; lowdensity lipoprotein (LDL) or an apoprotein of LDL; steroid; steroidderivative; hormone; hormone-mimic; lectin; drug; antibiotic; aptamer;DNA; RNA; lipid; an antibody; and an antibody-related polypeptide.

In an embodiment, the at least one photoactivatable molecule includes,but is not limited to, at least one of a: porphyrin; chlorin;bacteriochlorin; isbacteriochlorin; phthalocyanine; napthalocyanine;porphycene; porphycyanine; tetra-macrocyclic compound; poly-macrocycliccompound; pyropheo-phorbide; pentaphyrin; sapphyrin; texaphyrin; metalcomplex; tetrahydrochlorin; phonoxazine dye; phenothiazine;chaloorganapyrylium dye; rhodamine; fluorescene; azoporphyrin;benzochlorin; purpurin; chlorophyll; verdin; triarylmethane; angelicin;chalcogenapyrillium dye; chlorophyll; coumarin; cyanine; ceratindaunomycin; daunomycinone; 5-iminodauno-mycin; doxycycline; furosemide;gilvocarcin M; gilvocarcin V; hydroxy-chloroquine sulfate;lumidoxycycline; mefloquine hydrochloride; mequitazine; merbromin(mercurochrome); primaquine diphosphate; quinacrine dihydrochloride;quinine sulfate; tetracycline hydrochloride; flavins; alloxazine; flavinmononucleotide; 3-hydroxyflavone; limichrome; limiflavin;6-methylalloxazine; 7-methylalloxazine; 8-methylalloxazine;9-methylalloxazine; 1-methyl limichrome; methyl-2-methoxybenzoate;5-nitrosalicyclic acid; proflavine; and riboflavin; metallo-porphyrin;metallophthalocyanine; methylene blue derivative; naphthalmide;naphthalocyanine; pheophorbide; pheophytin; photosensitizer dimer andconjugate; phthalocyanine; porphycene; quinone; retinoid; rhodamine;thiophene; verdin; vitamin; and xanthene dye.

In an embodiment, the quencher molecule includes, but is not limited to,at least one of: 4-(4′-dimethylamino-phenylazo)benzoic acid (DABCYL);dabcyl succinimidyl ester; 4-(4′-dimethylamino-phenylazo)sulfonic(DABSYL); dabsyl succinimidyl ester; tetramethyl-rhodamaine (TAMRA);4-[(4-nitrophenyl)diazinyl]-phenylamine and4-[4-nitrophenyl)diazinyl]-naphthylamine; dabcylnitro-thiazole;6-(N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl]amino)hexanoic acid;6-carboxy-X-rhodamine (ROX);QSY-7;2-[4-(4-nitrophenylazo)-N-ethylphenyl-amino]ethanol (Disperse Red 1);2-[4-(2-chloro-4-nitrophenyl-azo)-N-ethylphenylamino]-ethanol (DisperseRed 13); tetrarhodamine isothiocyanate (TRITC); allophycocyanin;β-carotene; diarylrhodamine derivatives, QSY 7, QSY 9, QSY 21 dyes; QSY35 acetic acid succinimidyl ester; QSY 35 iodoacetamide; aliphaticmethylamine; napthalate; Reactive Red 4; and Malachite Green.

In an embodiment, the red blood cell includes, but is not limited to, atleast one of: a reticulocyte; a red blood cell; and a fused red bloodcell including a red blood cell autologous to a subject fused to one ormore allogeneic erythrocytes, liposomes or artificial vesicles.

In an embodiment, the at least one photoactivatable molecule is operablylinked to the quenching molecule and the target recognition moiety sothat the at least one photoactivatable molecule is quenched until thetarget recognition moiety is bound to the target molecule, whereupon thequenching molecule moves away from the photoactivatable molecule,enabling excitation of the photoactivatable molecule upon irradiationwith the light of a suitable wavelength.

In an embodiment, the red blood cell further comprises at least onetherapeutic agent. In an embodiment, the therapeutic agent includes, butis not limited to, at least one of: an antibiotic, an antiviral agent,an antifungal agent, an anti-parasitic agent, an antibody, anantibody-related polypeptide, a chemotherapeutic agent; a protein-basedpharmaceutical; and an RNA-based pharmaceutical. In an embodiment, theantibody is a monoclonal antibody, a polyclonal antibody, a fragmentthereof, including but not limited to a ScFv fragment, or a Fabfragment, or antibody-like molecules.

In an embodiment, the red blood cell is engineered to further compriseone or more of another target recognition moiety including: an antigen;ligand; receptor; one member of a specific binding pair; polyamide;peptide; carbohydrate; oligosaccharide; polysaccharide; low densitylipoprotein (LDL) or an apoprotein of LDL; steroid; steroid derivative;hormone; hormone-mimic; lectin; drug; antibiotic; aptamer; DNA; RNA;lipid; antibody; and an antibody-related polypeptide.

In an aspect, a method for administering a photodynamic therapy to atarget is described, the method, comprising the steps of: (i)administering to a subject in need thereof, a modified red blood cellthat preferentially associates with the target; and (ii) irradiating atleast a portion of the subject with light of a wavelength and totalfluence configured to produce a therapeutic effect.

In an embodiment, the target includes, but is not limited to, at leastone of a vascular endothelial tissue; a neovasculature tissue; aneovasculature tissue present in an eye; an abnormal vascular wall of atumor; a solid tumor; a tumor of a head; a tumor of a neck; a tumor ofan eye; a tumor of a gastrointestinal tract; a tumor of a liver, a tumorof a breast; a tumor of a prostate; a tumors of a lung; a nonsolidtumor; malignant cells of one of a hematopoietic tissue, a lymphoidtissue, skin tissue; lesions in a vascular system, a diseased bonemarrow; noncancerous hyperproliferative tissue; and diseased cells inwhich the disease is one of an autoimmune and an inflammatory disease.

In an embodiment, the target includes, but is not limited to, at leastone of: a bacteria; a virus; a fungi; and a parasite.

In an embodiment, the parasite includes, but is not limited to, at leastone of: trypanosomes; haemoprotozoa and parasites capable of causingmalaria; enteric and systemic cestodes; taeniid cestodes; entericcoccidians; enteric flagellate protozoa; filarial nematodes;gastrointestinal and systemic nematodes and hookworms.

In an aspect, the use of a modified blood cell is described, wherein theuse is in a treatment of a medical condition selected from the groupconsisting of: atherosclerosis; restenosis; cancer; cancer precurors;noncancerous hyperproliferative diseases; psoriasis; maculardegeneration; glaucoma; viral infection; bacterial infection; fungalinfection; and a parasitic infection.

In an aspect, a method for detecting a target in a biological sample isdescribed, the method comprising: (i) adding to the biological samplethe modified red blood cell that binds to the target; and (ii) detectingthe modified red blood cell.

In an embodiment, the target includes, but is not limited to, at leastone of a vascular endothelial tissue; a neovasculature tissue; aneovasculature tissue present in an eye; an abnormal vascular wall of atumor; a solid tumor; a tumor of a head; a tumor of a neck; a tumor ofan eye; a tumor of a gastrointestinal tract; a tumor of a liver, a tumorof a breast; a tumor of a prostate; a tumors of a lung; a nonsolidtumor; malignant cells of one of a hematopoietic tissue, a lymphoidtissue, skin tissue; lesions in a vascular system, a diseased bonemarrow; noncancerous hyperproliferative tissue; and diseased cells inwhich the disease is one of an autoimmune and an inflammatory disease.

In an embodiment, the bacteria includes, but is not limited to, at leastone of: Neisseria meningitides; Neisseria gonorrheoeae; Legionella;Vibrio cholerae; Streptococci; Staphylococcus aureus; Staphylococcusepidermidis; Pseudomonas aeruginosa; Corynobacteria diphtheriae;Clostridium spp.; Eschericia coli; Bacillus anthracis; Bartonellahenselae; Bartonella quintana; Coxiella burnetii; Chlamydia;Mycobacterium leprae; Salmonella; Shigella; Yersinia enterocolitica;Yersinia pseudotuberculosis; Legionella pneumophila; Mycobacteriumtuberculosis; Listeria monocytogenes; Mycoplasma spp.; Pseudomonasfluorescens; Vibrio cholerae; Haemophilus influenzae; Bacillusanthracis; Treponema pallidum; Leptospira; Borrelia; Corynebacteriumdiphtheriae; Francisella; Brucella melitensis; Campylobacter jejuni;Enterobacter; Proteus mirabilis; Proteus; and Klebsiella pneumoniae.

In an embodiment, the fungi includes, but is not limited to, at leastone of: Candida albicans; Candida glabrata; Aspergilus spp.; Torulopsisglabrata; Candida tropicalis; C. krusei; and C. parapsilosis.

In an embodiment, the virus includes, but is not limited to, at leastone of: adenovirus; coxsackievirus; hepatitis A virus; poliovirus;epstein-barr virus; herpes simplex type 1; herpes simplex type 2; humancytomegalovirus; human herpes virus type 8; varicella-zoster virus;hepatitis B virus; a hepatitis C virus; human immunodeficiency virus(HIV); influenza virus; measles virus; mumps virus; parainfluenza virus;respiratory syncytial virus; papillomavirus; rabies virus; and Rubellavirus.

In an embodiment, the parasite includes, but is not limited to, at leastone of trypanosomes; haemoprotozoa; parasites from the genus Plasmodium,including but not limited to, Plasmodium falciparum, Plasmodium vivax,Plasmodium ovale, Plasmodium malaria; enteric and systemic cestodes;taeniid cestodes; enteric coccidians; enteric flagellate protozoa;filarial nematodes; gastrointestinal and systemic nematodes andhookworms.

In an embodiment, the biological sample includes, but is not limited to,at least one of blood, urine, saliva, tears, synovial fluid, sweat,interstitial fluid, sperm, cerebrospinal fluid, ascites fluid, tumortissue biopsy; tissue biopsy; and circulating tumor cells.

In an aspect, a method of generating an image of a target tissue or amodified red blood cell in a subject is described, the methodcomprising: (i) administering to the subject the modified red bloodcell; and (ii) generating an image of at least a portion of the subjectto which the modified red blood cell has preferentially associated.

In an aspect, a kit to treat a medical condition using a photodynamictherapy is described, the method comprising the modified red blood celland instructions for providing photodynamic therapy. In an embodiment,the medical condition includes, but is not limited to, at least one of:atherosclerosis; restenosis; cancer; cancer precurors; noncanceroushyperproliferative diseases; psoriasis; macular degeneration; glaucoma;viruses; bacterial infection; fungal infection; and a parasiticinfection.

In an aspect, a kit to specifically label a cell or tissue is described,the kit comprising: the modified red blood cell comprising at least onetarget recognition moiety directed to the specific cell or tissue; andinstructions teaching a method of imaging the modified red blood cell.

In an aspect, a method of making an isolated modified red blood cell isdescribed, the method comprising providing a red blood cell; contactingthe red blood cell with a one or more target-binding agents comprising atarget recognition moiety, a photoactivatable molecule, and a quenchermolecule, under conditions wherein the one or more target-binding agentis associated with the red blood cell to produce a modified red bloodcell; and isolating the modified red blood cell from the one or moretarget-binding agent which is not associated with the modified red bloodcell, thereby making an isolated modified red blood cell.

In an embodiment, the step of providing the red blood cell comprisesdifferentiating erythrocytes ex vivo from a stem cell or a reticulocyte.In an embodiment, the stem cells are hematopoietic stem cells. In anembodiment, the red blood cell is autologous to the subject. In anembodiment, the red blood cell may be allogeneic to the subject. In anembodiment, the allogeneic red blood cell includes one or more bloodtype specific erythrocytes or one or more universal donor erythrocytes.In an embodiment, the red blood cells are erythrocytes fused betweenerythrocytes autologous to the subject and one or more allogeneicerythrocytes, liposomes, or artificial vesicles. In an embodiment, thered blood cell may be engineered to produce a protein-basedpharmaceutical or an RNA-based pharmaceutical.

In an embodiment, the method further comprises the step of loading themodified red blood cell with a therapeutic agent. In an embodiment, thetherapeutic agent includes, but is not limited to, at least one of: anantibiotic, an antiviral agent, an antifungal agent, an anti-parasiticagent, an antibody, an antibody-related polypeptide; a chemotherapeuticagent; a protein-based pharmaceutical; and an RNA-based pharmaceutical.

The foregoing summary is illustrative only and is not intended to be inany way limiting. In addition to the illustrative aspects, embodiments,and features described above, further aspects, embodiments, and featureswill become apparent by reference to the drawings and the followingdetailed description.

BREEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram illustrating the activation of atarget-binding agent upon binding to a target molecule and exposure tolight of a suitable wavelength and power.

FIG. 2 is a schematic diagram illustrating the interaction of a modifiedred blood cell with a target cell. The target-binding agent is activatedupon binding to the target cell and singlet oxygen radical species isgenerated upon exposure to electromagnetic radiation of a suitablewavelength and power.

FIG. 3 is a schematic diagram illustrating the interaction of multipletarget-binding agents with a target cell. The target-binding agents areactivated upon binding to the target cell.

FIG. 4 is a schematic diagram illustrating the interaction of apopulation of modified red blood cells with a population of targetcells. The target-binding agent is activated upon binding to the targetcell.

FIG. 5 is a schematic diagram illustrating the interaction of a modifiedred blood cell with a target cell. Exposure of the activatedtarget-binding agent to the electromagnetic radiation of a suitablewavelength and power produces a singlet oxygen radical molecule which,in turn, results in damage or death to the target cell.

FIG. 6 is a schematic diagram illustrating the interaction of a modifiedred blood cell with a target cell. The target-binding agent is activatedupon binding of the target recognition moiety to the target molecule.Exposure of the activated target-binding agent to electromagneticradiation of a suitable wavelength produces a singlet oxygen radicalmolecule which results in lysis of the modified red blood cell andrelease of one or more therapeutic agents.

FIG. 7 is a schematic diagram illustrating the interaction of a modifiedred blood cell with a target cell. The modified red blood cell comprisesmultiple target recognition moieties on its surface.

FIG. 8 is a schematic diagram illustrating the interaction of a modifiedred blood cell with a target cell. The target cell becomes internalizedwithin the modified red blood cell, thereby activating the targetrecognition moiety of the target-binding agent.

FIG. 9 is a schematic diagram illustrating the interaction of a modifiedred blood with a target cell. The modified red blood cell becomesinternalized into the target cell, where target molecules become boundto the target-binding agent, thereby activating the target recognitionmoiety of the target-binding agent.

FIG. 10 is a schematic diagram illustrating the interaction of amodified red blood cell with a target cell. The modified red blood cellbecomes internalized into the target cell, where target molecules becomebound to the target-binding agent, thereby activating the targetrecognition moiety of the target-binding agent. The activatedtarget-binding agent produces a singlet oxygen radical molecule when itis exposed to electromagnetic radiation of a suitable wavelength andpower, which results in lysis of the modified red blood cell and releaseof one or more therapeutic agents.

DETAILED DESCRIPTION

Described herein are modified red blood cells. More particularlydescribed herein are compositions comprising a red blood cell associatedwith a target recognition moiety and a fusion protein. In an embodiment,the modified red blood cell may comprise a photoactivatable molecule anda quencher molecule, wherein the target-binding agent emits at least onesinglet oxygen radical molecule upon exposure to electromagneticradiation (e.g., light) of a suitable wavelength when the target-bindingagent is bound to a target molecule. Also described are targeteddelivery of imaging agents, drugs, and peptide and proteinpharmaceuticals using modified red blood cells. Processes for preparingthe modified red blood cells, pharmaceutical and diagnostic compositionscontaining the same and methods of diagnosis and treatment involving themodified red blood cells are described. The specific compositions andmethods described herein are intended as merely illustrative of theirmore general counterparts.

In this dislcosure, many conventional techniques in molecular biology,protein biochemistry, cell biology, immunology, microbiology andrecombinant DNA are described or referenced. These techniques arewell-known and are explained in, e.g., Current Protocols in MolecularBiology, Vols. I-III, Ausubel, Ed. (1997); Sambrook et al., MolecularCloning. A Laboratory Manual, Second Ed. (Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989); DNA Cloning: A PracticalApproach, Vols. I and II, Glover, Ed. (1985); Oligonuchotide Synthesis,Gait, Ed. (1984); Nucleic Acid Hybridization, Hames & Higgins, Eds.(1985); Transcription and Translation, Hames & Higgins, Eds. (1984);Animal Cell Culture, Freshney, Ed. (1986); Immobilized Cells and Enzymes(IRL Press, 1986); Perbal, A Practical Guide to Molecular Cloning; theseries, Meth. Enzymol., (Academic Press, Inc., 1984); Gene TransferVectors for Mammalian Cells, Miller & Calos, Eds. (Cold Spring HarborLaboratory, NY, 1987); and Meth. Enzymol., Vols. 154 and 155, Wu &Grossman, and Wu, Eds., respectively. and Strachan & Read, HumanMolecular Genetics, Second Edition. (John Wiley and Sons, Inc., NY,1999)).

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural referents unless the contentclearly dictates otherwise. For example, reference to “a cell” include asingle cell or may include a combination of two or more cells, and thelike. Generally, the nomenclature used herein and the laboratoryprocedures in cell culture, molecular genetics, organic chemistry,analytical chemistry and nucleic acid chemistry and hybridizationdescribed below are those well known and commonly employed in the art.

Units, prefixes, and symbols may be denoted in their accepted SI form.Unless otherwise indicated, nucleic acids are written left to right in5′ to 3′ orientation; amino acid sequences are written left to right inamino to carboxy orientation. Amino acids may be referred to herein byeither their commonly known three letter symbols or by the one-lettersymbols recommended by the IUPAC-IUBMB Nomenclature Commission.Nucleotides, likewise, may be referred to by their commonly acceptedsingle-letter codes.

All references cited herein are incorporated herein by reference to theextent not inconsistent with the instant disclosure and for all purposesto the same extent as if each individual publication, patent, or patentapplication was specifically and individually incorporated by reference.

The administration of an agent or drug to a subject or subject includesany route of introducing or delivering to a subject a compound toperform its intended function. Administration can be carried out by anysuitable route, including orally, intranasally, parenterally(intravenously, intramuscularly, intraperitoneally, or subcutaneously),rectally, or topically. Administration includes self-administration andthe administration by another.

An antibody includes a polypeptide comprising a framework region from animmunoglobulin gene or fragments thereof that specifically binds andrecognizes an antigen. Use of the term antibody is meant to includewhole antibodies, including single-chain antibodies, antibody fragments,and antibody-related polypeptides.

Antibody includes bispecific antibodies and multispecific antibodies solong as they exhibit the desired biological activity or function.

An antibody-related polypeptide includes antigen-binding antibodyfragments, including single-chain antibodies, that can comprise thevariable region(s) alone, or in combination, with all or part of thefollowing polypeptide elements: hinge region, CH₁, CH₂, and CH₃ domainsof an antibody molecule. Also included are any combinations of variableregion(s) and hinge region, CH₁, CH₂, and CH₃ domains. Antibody-relatedmolecules useful as binding agents include, e.g., but are not limitedto, Fab, Fab′ and F(ab′)₂, Fd, single-chain Fvs (scFv), single-chainantibodies, disulfide-linked Fvs (sdFv) and fragments comprising eithera V_(L) or V_(H) domain. Examples include: (i) a Fab fragment, amonovalent fragment consisting of the V_(L), V_(H), C_(L) and C_(H),domains; (ii) a F(ab′)₂ fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; (iii) a Fdfragment consisting of the V_(H) and CH₁ domains; (iv) a Fv fragmentconsisting of the V_(L) and V_(H) domains of a single arm of anantibody, (v) a dAb fragment (Ward et al., Nature 341: 544-546 (1989)),which consists of a V_(H) domain; and (vi) an isolated complementaritydetermining region (CDR). As such antibody fragments may comprise aportion of a full length antibody, generally the antigen binding orvariable region thereof. Examples of antibody fragments include, but arenot limited to, Fab, Fab′, F(ab′)₂, and Fv fragments; diabodies; linearantibodies; single-chain antibody molecules; and multispecificantibodies formed from antibody fragments. Single-chain antibodymolecules may comprise a polymer with a number of individual molecules,for example, dimmer, trimer or other polymers.

A biological sample includes sample material derived from or contactedby living cells. The term “biological sample” is intended to includetissues, cells and biological fluids isolated from a subject, as well astissues, cells and fluids present within a subject. Biological samplesinclude, e.g., but are not limited to, whole blood, plasma, semen,saliva, tears, urine, fecal material, sweat, buccal, skin, cerebrospinalfluid, and hair. Biological samples can also be obtained from biopsiesof internal organs or from cancers. Biological samples can be obtainedfrom subjects for diagnosis or research or can be obtained fromundiseased individuals, as controls or for basic research.

A antineoplastic agent includes a chemical compound that can be usedeffectively to treat a neoplastic cell.

An effective amount or pharmaceutically effective amount ortherapeutically effective amount of a composition, includes a quantityof material sufficient to reasonably achieve a desired therapeuticand/or prophylactic effect. For example, it may include an amount thatresults in the prevention of, treatment of, or a decrease in, thesymptoms associated with a disease or condition that is being treated,e.g., the diseases or medical conditions associated with a targetpolypeptide. The amount of a therapeutic composition administered to thesubject will depend on the type and severity of the disease and on thecharacteristics of the individual, such as general health, age, sex,body weight and tolerance to drugs. It will also depend on the degree,severity and type of disease. The skilled artisan will be able todetermine appropriate dosages depending on these and other factors. Thecompositions can also be administered in combination with one or moreadditional therapeutic compounds.

Electromagnetic radiation of a suitable wavelength includes one or morefrequencies of electromagnetic radiation having one or morecharacteristics that taken as a whole are not considered unduly harmfulto the subject. In illustrative non-limiting examples, suchelectromagnetic energy may include frequencies of optical light,optionally including visible light (detected by the human eye betweenapproximately 400 nm and 700 nm) as well as infrared (longer than 700nm) and limited spectral regions of ultraviolet light, such as UVA light(between approximately 320 nm and 400 nm). Electromagnetic energyincludes, but is not limited to, single photon electromagnetic energy,two photon electromagnetic energy, multiple wavelength electromagneticenergy, and extended-spectrum electromagnetic energy.

An epitope includes any segment on an antigen to which an antibody orother ligand or binding molecule binds. An epitope may consist ofchemically active surface groupings of molecules such as amino acids orsugar side chains and usually have specific three dimensional structuralcharacteristics, as well as specific charge characteristics.

A monoclonal antibody includes an antibody obtained from a population ofsubstantially homogeneous antibodies, i.e., the individual antibodiescomprising the population are identical except for possible naturallyoccurring mutations that may be present in minor amounts. For example, amonoclonal antibody can be an antibody that is derived from a singleclone, including any eukaryotic, prokaryotic, or phage clone, and notthe method by which it is produced. A monoclonal antibody compositiondisplays a single binding specificity and affinity for a particularepitope. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen.

A non-target tissue includes tissue of the subject which are notintended to be impaired or destroyed by the treatment method. Thesenon-target tissues include but are not limited to healthy blood cells,and other normal tissue, not otherwise identified to be targeted.

A photoactivatable molecule or photosensitizing agent includes achemical compound that upon exposure to photoactivating electromagneticradiation is activated to release a singlet oxygen molecule. In anembodiment, the photoactivatable molecule itself, or some other species,is converted into a cytotoxic form, whereby target cells are killed ortheir proliferative potential diminished. Thus, photoactivatablemolecule may exert their effects by a variety of mechanisms, directly orindirectly. For example, certain photoactivatable molecules become toxicwhen activated by light, for example by generating toxic species, e.g.,oxidizing agents such as singlet oxygen or oxygen-derived free radicals,which are extremely destructive to cellular material and biomoleculessuch as lipids, proteins and nucleic acids. Porphyrins are ofphotosensitizing agents that act by generation of toxic oxygen species.Typically, the chemical compound is nontoxic to the animal to which itis administered or is capable of being formulated in a nontoxiccomposition, and the chemical compound in its photodegraded form is alsonontoxic. A listing of representative photosensitive chemicals may befound in Kreimer-Bimbaurn, Sem. Hematol. 26:157-73 (1989).

A quencher, quencher molecule, or quenching molecule includes a moietycapable of preventing activation of the photoactivatable molecule whenthe target-binding agent is not bound to the target. Alternatively, thequencher may be capable of preventing the release of singlet oxygen fromthe target-binding agent when the target-binding agent is not bound tothe target. In a suitable embodiment, the photoactivatable molecule is aporphyrin, and the quencher includes one or more suitable functionalgroups that coordinate to the axial position of the metal coordinatedwithin the photoactivatable molecule. The target recognition moiety ispositioned in the agent in such a way that the interaction of the targetrecognition moiety with the target disrupts the association of the axialligand to the metal, releasing the quenching agent and allowing theporphyrin or porphyrin derivative tetrapyrrole to be activated whenirradiated.

A subject includes, but is not limited to, a mammal, such as a human,but can also be an animal, e.g., domestic animals (e.g., dogs, cats andthe like), farm animals (e.g., cows, sheep, pigs, horses and the like)and laboratory animals (e.g., monkey, rats, mice, rabbits, guinea pigsand the like).

A target includes the object that is intended to be detected, diagnosed,impaired or destroyed by the methods provided herein, and includestarget cells, target tissues, and target compositions. Target cells arecells in target tissue, and the target tissue includes, but is notlimited to, vascular endothelial tissue, abnormal vascular walls oftumors, solid tumors such as (but not limited to) tumors of the head andneck, tumors of the eye, tumors of the gastrointestinal tract, tumors ofthe liver, tumors of the breast, tumors of the prostate, tumors of thelung, nonsolid tumors and malignant cells of the hematopoietic andlymphoid tissue, neovascular tissue, other lesions in the vascularsystem, bone marrow, and tissue or cells related to autoimmune disease.Also included among target cells are cells undergoing substantially morerapid division as compared to non target cells, as well as pathogenssuch as bacteria, fungi, viruses, and parasites.

A target recognition moiety includes a molecule that is configured tospecifically bind with a target. In an embodiment, the targetrecognition moiety is a member of a specific binding pair, e.g., anantigen; ligand; receptor; polyamide; peptide; carbohydrate;oligosaccharide; polysaccharide; low density lipoprotein (LDL) or anapoprotein of LDL; steroid; steroid derivative; hormone; hormone-mimic;lectin; drug; antibiotic; aptamer; DNA; RNA; lipid; or an antibody orantibody-related polypeptide.

A therapeutic agent includes a compound or molecule that, when presentin an effective amount, produces a desired therapeutic effect on asubject in need thereof.

Compositions

In an aspect, modified red blood cells are described that are configuredto target specific cells or tissues. In an embodiment, the modified redblood cells comprise at least one target-binding agent including atarget recognition moiety and one or more fusion molecules. The modifiedred blood cells may be used as a vehicle to deliver myriad agents totargets in vivo. In an embodiment, the modified red blood cells includea photoactivatable molecule that is activated upon binding of a targetrecognition moiety to a target molecule. When the photoactivatablemolecule is exposed to photoactivating light, one or more singlet oxygenradical molecules is produced, which may kill or damage target cells ortissues directly or indirectly. In this section, the components of themodified red blood cells are described.

I. Preparation of Modified Red Blood Cells

A. Preparation of Red Blood Cells

1. Isolation of Red Blood Cells

Mature red blood cells for use in generating the modified red bloodcells may be isolated using various methods such as, for example, a cellwasher, a continuous flow cell separator, density gradient separation,fluorescence-activated cell sorting (FACS), Miltenyi immunomagneticdepletion (MACS), or a combination of these methods (See, e.g., van derBerg et al., Clin. Chem. 33:1081-1082(1987); Bar-Zvi et al., J. Biol.Chem. 262:17719-17723 (1987); Goodman et al., Exp. Biol. Med.232:1470-1476 (2007)).

Red blood cells may be isolated from whole blood by simplecentrifugation (See, e.g., van der Berg et al., Clin. Chem. 33:1081-1082(1987)). For example, EDTA-anticoagulated whole blood may be centrifugedat 800×g for 10 min at 4° C. The platelet-rich plasma and buffy coat areremoved and the red blood cells are washed three times with isotonicsaline solution (NaCl, 9 g/L).

Alternatively, red blood cells may be isolated using density gradientcentrifugation with various separation mediums such as, for example,Ficoll, Hypaque, Histopaque, Percoll, Sigmacell, or combinationsthereof. For example, a volume of Histopaque-1077 is layered on top ofan equal volume of Histopaque-1119. EDTA-anticoagulated whole blooddiluted 1:1 in an equal volume of isotonic saline solution (NaCl, 9 g/L)is layered on top of the Histopaque and the sample is centrifuged at700×g for 30 min at room temperature. Under these conditions,granulocytes migrate to the 1077/1119 interface, lymphocytes, othermononuclear cells and platelets remain at the plasma/1077 interface, andthe red blood cells are pelleted. The red blood cells are washed twicewith isotonic saline solution.

Alternatively, red blood cells may be isolated by centrifugation using aPercoll step gradient (See, e.g., Bar-Zvi et al., J. Biol. Chem.262:17719-17723 (1987)). As such, fresh blood is mixed with ananticoagulant solution containing 75 mM sodium citrate and 38 mM citricacid and the cells washed briefly in Hepes-buffered saline. Leukocytesand platelets are removed by adsorption with a mixture of α-celluloseand Sigmacell (1:1). The red blood cells are further isolated fromreticulocytes and residual white blood cells by centrifugation through a45/75% Percoll step gradient for 10 min at 2500 rpm in a Sorvall SS34rotor. The red blood cells are recovered in the pellet whilereticulocytes band at the 45/75% interface and the remaining white bloodcells band at the 0/45% interface. The Percoll is removed from the redblood cells by several washes in Hepes-buffered saline. Other materialsthat may be used to generate density gradients for isolation of redblood cells include OptiPrep™, a 60% solution of iodixanol in water(from Axis-Shield, Dundee, Scotland).

Red blood cells may be separated from reticulocytes, for example, usingflow cytometry (See, e.g., Goodman el al., Exp. Biol. Med. 232:1470-1476(2007)). In this instance, whole blood is centrifuged (550×g, 20 min,25° C.) to separate cells from plasma. The cell pellet is resuspended inphosphate buffered saline solution and further fractionated onFicoll-Paque (1.077 density), for example, by centrifugation (400×g, 30min, 25° C.) to separate the red blood cells from the white blood cells.The resulting cell pellet is resuspended in RPMI supplemented with 10%fetal bovine serum and sorted on a FACS instrument such as, for example,a Becton Dickinson FACSCalibur (BD Biosciences, Franklin Lakes, N.J.,USA) based on size and granularity.

Red blood cells may be isolated by immunomagnetic depletion (See, e.g.,Goodman, el al., (2007) Exp. Biol. Med. 232:1470-1476). In thisinstance, magnetic beads with cell-type specific antibodies are used toeliminate non-red blood cells. For example, red blood cells are isolatedfrom the majority of other blood components using a density gradient asdescribed above followed by immunomagnetic depletion of any residualreticulocytes. The cells are pre-treated with human antibody serum for20 min at 25° C. and then treated antibodies against reticulocytespecific antigens such as, for example, CD71 and CD36. The antibodiesmay be directly attached to magnetic beads or conjugated to PE, forexample, to which magnetic beads with anti-PE antibody will react. Assuch, the antibody-magnetic bead complex is able to selectively extractresidual reticulocytes, for example, from the red blood cell population.

Red blood cells may also be isolated using apheresis. The process ofapheresis involves removal of whole blood from a patient or donor,separation of blood components using centrifugation or cell sorting,withdrawal of one or more of the separated portions, and transfusion ofremaining components back into the patient or donor. A number ofinstruments are currently in use for this purpose such as for examplethe Amicus and Alyx instruments from Baxter (Deerfield, Ill., USA), theTrima Accel instrument from Gambro BCT (Lakewood, Colo., USA), and theMCS+9000 instrument from Haemonetics (Braintree, Mass., USA). Additionalpurification methods, such as those described above, may be necessary toachieve the appropriate degree of red blood cell purity.

2. Allogenic and Autologous Modified Red Blood Cells

In an embodiment, the modified red blood cells are autologous and/orallogeneic to the subject. In an embodiment, erythrocytes allogeneic tothe subject include one or more of one or more blood type specificerythrocytes or one or more universal donor erythrocytes. In anembodiment, the modified red blood cells are fusion erythrocytes betweenerythrocytes autologous to the subject and one or more allogeneicerythrocytes, liposomes, and/or artificial vesicles.

For autologous transfusion, red blood cells, reticulocytes orhematopoietic stem cells from an individual are isolated and modified bymethods described herein and retransfused into the individual.

For allogeneic transfusions, red blood cells, reticulocytes orhematopoietic stem cells are isolated from a donor, modified by methodsdescribed herein and transfused into another individual. In the instancewhere allogeneic cells are used for transfusion, care needs to be takento use a compatible ABO blood group to prevent an acute intravascularhemolytic transfusion reaction. The latter is characterized bycomplement activation and lysis of incompatible red blood cells. The ABOblood types are defined based on the presence or absence of the bloodtype antigens A and B, monosaccharide carbohydrate structures that arefound at the termini of oligosaccharide chains associated withglycoproteins and glycolipids on the surface of the red blood cells(reviewed in Liu et al., Nat. Biotech. 25:454-464 (2007)). Group O redblood cells lack either of these antigenic monosaccharide structures.

Individuals with group A red blood cells have naturally occurringantibodies to group B red blood cells whereas individuals with group Bred blood cells have antibodies to group A red blood cells. Blood groupAB individuals have neither antibody and blood group O individuals haveboth. Individuals with either anti-A and/or anti-B antibodies cannotreceive a transfusion of blood containing the corresponding antigen.Because group O red blood cells contain neither A nor B antigens, theycan be safely transfused into recipients of any ABO blood group, i.e.,group A, B, AB, or O recipients. As such, group O red blood cells areconsidered “universal” and may be used in all blood transfusions. Incontrast, group A red blood cells may be given to group A and ABrecipients, group B red blood cells may be given to group B and ABrecipients, and group AB red blood cells may only be given to ABrecipients. As such, the modified red blood cells with an activatablemolecular marker are matched for compatibility with the recipient.

In some instances, it may be beneficial to convert a non-group Omodified red blood cell to a universal blood type. Enzymatic removal ofthe immunodominant monosaccharides on the surface of group A and group Bred blood cells is one approach to generating a group O-like red bloodcell population (See, e.g., Liu et al., Nat. Biotech. 25:454-464(2007)). Group B red blood cells may be converted using anα-galactosidase derived from green coffee beans, for example.Alternatively, α-N-acetylgalactosaminidase and α-galactosidase enzymaticactivities derived from E. meningosepticum bacteria may be used torespectively remove the immunodominant A and B antigens (Liu et al.,Nat. Biotech. 25:454-464 (2007)). As such, packed red blood cellsisolated as described above, are incubated in 200 mM glycine (pH 6.8)and 3 mM NaCl in the presence of either α-N-acetylgalactosaminidase andα-galactosidase (˜300 μg/ml packed red blood cells) for 60 min at 26° C.After treatment, the red blood cells are washed by 3-4 rinses in salinewith centrifugation and ABO-typed according to standard blood bankingtechniques.

3. Derivation of Erythrocytes from Reticulocytes

In an embodiment, the red blood cells are differentiated ex vivo and/orin vivo from one or more reticulocytes. Modified reticulocytes may beused to generate mature red blood cells with monitoring and/ortherapeutic properties. Reticulocytes are immature red blood cells andcompose approximately 1% of the red blood cells in the human body.Reticulocytes develop and mature in the bone marrow. Once released intocirculation, reticulocytes rapidly undergo terminal differentiation tomature red blood cells. Like mature red blood cells, reticulocytes donot have a cell nucleus. Unlike mature red blood cells, reticulocytesmaintain the ability to perform protein synthesis. As such, theintroduction of foreign messenger RNA (mRNA) into reticulocytes mayfacilitate synthesis and expression of exogenous proteins and/orpeptides.

Reticulocytes of varying age may be isolated from peripheral blood basedon the differences in cell density as the reticulocytes mature. As such,reticulocytes may be isolated from peripheral blood using differentialcentrifugation through various density gradients. For example, Percollgradients may be used to isolate reticulocytes (See, e.g., Noble el al.,Blood 74:475-481 (1989)). Sterile isotonic Percoll solutions of density1.096 and 1.058 g/ml are made by diluting Percoll (Sigma-Aldrich, SaintLouis, Mo., USA) to a final concentration of 10 mM triethanolamine, 117mM NaCl, 5 mM glucose, and 1.5 mg/ml bovine serum albumin (BSA). Thesesolutions have an osmolarity between 295 and 310 mOsm. Five milliliters,for example, of the first Percoll solution (density 1.096) is added to asterile 15 ml conical centrifuge tube. Two milliliters, for example, ofthe second Percoll solution (density 1.058) is layered over the higherdensity first Percoll solution. Two to four milliliters of whole bloodare layered on top of the tube. The tube is centrifuged at 250×g for 30min in a refrigerated centrifuge with swing-out tube holders.Reticulocytes and some white cells migrate to the interface between thetwo Percoll layers. The cells at the interface are transferred to a newtube and washed twice with phosphate buffered saline (PBS) with 5 mMglucose, 0.03 mM sodium azide and 1 mg/ml BSA. Residual white bloodcells are removed by chromatography in PBS over a size exclusion column.

Alternatively, reticulocytes may be isolated by positive selection usingan immunomagnetic separation approach (See, e.g., Brun et al., Blood76:2397-2403 (1990)). This approach takes advantage of the large numberof transferrin receptors that are expressed on the surface ofreticulocytes relative to erythrocytes prior to maturation. As such,magnetic beads coated with an antibody to the transferrin receptor maybe used to selectively isolate reticulocytes from a mixed red cellpopulation. Antibodies to the transferrin receptor of a variety ofmammalian species, including human, are available from commercialsources (e.g., Affinity BioReagents, Golden, Colo., USA; Sigma-Aldrich,Saint Louis, Mo., USA). The transferrin antibody may be directly linkedto the magnetic beads. Alternatively, the transferrin antibody may beindirectly linked to the magnetic beads via a secondary antibody. Forexample, mouse monoclonal antibody 10D2 (Affinity BioReagents, Golden,Colo., USA) against human transferrin may be mixed with immunomagneticbeads coated with a sheep anti-mouse immunoglobulin G (Dynal/Invitrogen,Carlsbad, Calif., USA). The immunomagnetic beads are then incubated witha leukocyte-depleted red blood cell (RBC) fraction. The beads and RBCsare incubated at 22° C. with gentle mixing for 60-90 min followed byisolation of the beads with attached reticulocytes using a magneticfield. The isolated reticulocytes may be removed from the magnetic beadsusing, for example, DETACHaBEAD® solution (from Invitrogen, Carlsbad,Calif., USA). Alternatively, reticulocytes may be isolated from in vitrogrowth and maturation of CD34+ hematopoietic stem cells using themethods described below.

In general, the purity of the isolated reticulocytes may be assessedusing microscopy in that reticulocytes are characterized by a reticular(mesh-like) network of ribosomal RNA that becomes visible under amicroscope with certain stains such as new methylene blue or brilliantcresyl blue. Alternatively, analysis of creatine and hemoglobin Aiccontent and pyruvate kinase, aspartate aminotransferase, andporphobilinogen deaminase enzyme activity may be used to assessproperties of the isolated reticulocytes relative to mature erythrocytes(See, e.g., Brun et al., Blood 76:2397-2403 (1990)). For example, theactivity of porphobilinogen deaminase is nearly 9 fold higher whereasthe hemoglobin Aic content is nearly 10 fold less in reticulocytesrelative to mature erythrocytes.

Modified reticulocytes may be transfused into an animal and allowed todifferentiate into mature erythrocytes in vivo. Alternatively, modifiedreticulocytes may be differentiated into mature erythrocytes in vitroprior to transfusion. Maturation of reticulocytes in vitro may becarried out over several days using standard cell culture methods (See,e.g., Noble et al., Blood 74:475-481 (1998)). For example, isolatedreticulocytes are cultured for 3-5 days at 37° C. in Alpha-minimumessential medium (MEM) supplemented with 25 mM HEPES, 20 mg/dL glucose,5% fetal bovine serum, 100 U/ml penicillin, and 0.1 mg/ml streptomycin,pH 7.5 at which time several assays may be done to assess maturation.For example, new methylene blue staining in combination with microscopymay be used to assess decline in the RNA-derived reticular network.Alternatively, the decline in the transferrin receptor expression as afunction of maturation may be monitored using transferrin labeled, forexample, with ¹²⁵I, or FITC (Noble et al., Blood 74:475-481 (1998)). Insome instances, the analysis of creatine and hemoglobin Aic content andpyruvate kinase, aspartate aminotransferase, and porphobilinogendeaminase enzyme activity may be used to assess maturation as describedherein.

4. Differentiation of Red Blood Cells from Hematopoeitic Stem Cells

In an embodiment, the red blood cells are differentiated ex vivo and/orin vivo from one or more stem cells. In an embodiment, the one or morestem cells are one or more hematopoietic stem cells.

Red blood cells for use in generating a one or more modified red bloodcells may be derived from hematopoietic stem cells. Hematopoietic stemcells give rise to all of the blood cell types found in mammalian bloodincluding myeloid (monocytes and macrophages, neutorphils, basophils,eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells)and lymphoid lineages (T-cells, B-cells, NK-cells). Hematopoietic stemcells may be isolated from the bone marrow of adult bones including, forexample, femur, hip, rib, or sternum bones. Cells may be obtaineddirectly from the hip, for example, by removal of cells from the bonemarrow using aspiration with a needle and syringe. Alternatively,hematopoietic stem cells may be isolated from normal peripheral bloodfollowing pre-treatment with cytokines such as, for example, granulocytecolony stimulating factor (G-CSF). G-CSF mobilizes the release of cellsfrom the bone marrow compartment into the peripheral circulation. Othersources of hematopoietic stem cells include umbilical cord blood andplacenta.

Isolated hematopoietic stem cells may be cultured, expanded anddifferentiated ex vivo. For example, hematopoietic stem cells isolatedfrom bone marrow, cytokine-stimulated peripheral blood or umbilical cordblood may be expanded and differentiated ex vivo into matureerythrocytes (Giarratana et al., Nature Biotech. 23:69-74 (2005); U.S.Patent Application 2007/0218552). As such, CD34+ cells are isolated frombone marrow or peripheral or cord blood using, for example, magneticmicrobead selection and Mini-MACS columns (Miltenyi Biotech). The cellsare subsequently cultured in modified serum-free medium supplementedwith 1% bovine serum albumin (BSA), 120 μg/ml iron-saturated humantransferrin, 900 ng/ml ferrous sulfate, 90 ng/ml ferric nitrate and 10μg/ml insulin and maintained at 37° C. in 5% carbon dioxide in air.Expansion and differentiation of the cell culture may occur in multiplesteps. For example, in the initial growth step following isolation, thecells may be expanded in the medium described herein in the presence ofmultiple growth factors including, for example, hydrocortisone, stemcell factor, IL-3, and erythropoietin. In the second stage, the cellsmay be co-cultured, for example, on an adherent stromal layer in thepresence of erythropoietin. In a third stage, the cells may be culturedon an adherent stromal layer in culture medium in the absence ofexogenous factors. The adherent stromal layer may be murine MS-5 stromalcells, for example. Alternatively, the adherent stromal layer may bemesenchymal stromal cells derived from adult bone marrow. The adherentstromal cells may be maintained in RPMI supplemented with 10% fetal calfserum, for example.

In some instances, it may be desirable to expand and partiallydifferentiate the CD34+ hematopoietic stem cells in vitro and to allowterminal differentiation into mature erythrocytes to occur in vivo (See,e.g., Neildez-Nguyen et al., Nature Biotech. 20:467-472 (2002)). Assuch, isolated CD34+ hematopoietic stem cells may be expanded in vitroin the absence of the adherent stromal cell layer in medium containingvarious factors including, for example, Flt3 ligand, stem cell factor,thrombopoietin, erythropoietin, and insulin growth factor. The resultingerythroid precursor cells, as judged by surface expression of CD36 andGPA, may be transfused into an animal where upon terminaldifferentiation to mature erythrocytes is allowed to occur.

Various assays may be performed to confirm the ex vivo differentiationof cultured hematopoietic stem cells into reticulocytes anderythrocytes, including, for example, microscopy, hematology, flowcytometry, deformability measurements, enzyme activities, and hemoglobinanalysis and functional properties (Giarratana et al., Nature Biotech.23:69-74 (2005)). The phenotype of cultured hematopoietic stem cells maybe assessed using microscopy of cells stained, for example, with CresylBrilliant blue. Reticulocytes, for example, exhibit a reticular networkof ribosomal RNA under these staining conditions whereas erythrocytesare devoid of staining. Enucleated cells may also be monitored forstandard hematological variables including mean corpuscular volume (MCV;fl), mean corpuscular hemoglobin concentration (MCHC; %) and meancorpuscular hemoglobin (MCH; pg/cell) using, for example, an XE2100automat (Sysmex, Roche Diagnostics).

For the deformability measurements, for example, presumptivereticulocytes may be separated from nucleated cells on day 15 ofculture, for example, by passage through a deleukocyting filter (e.g.,Leucolab LCG2, Macopharma) and subsequently assayed using ektacytometry.As such, the enucleated cells are suspended in 4% polyvinylpyrrolidonesolution and then exposed to an increasing osmotic gradient from 60 to450 mosM, for example. Changes in the laser diffraction pattern(deformability index) of the cells are recorded as a function ofosmolarity, to assess the dynamic deformability of the cell membrane.The maximum deformability index achieved at a physiologically relevantosmolarity is related to the mean surface area of red blood cells.

Alternatively, assays of hemoglobin may be used to assess the phenotypeof differentiated cells (Giarratana et al., Nature Biotech. 23:69-74(2005)). For example, high performance liquid chromatography (HPLC)using a Bio-Rad Variant II Hb analyzer (Bio-Rad Laboratories) may beused to assess the percentage of various hemoglobin fractions. Oxygenequilibrium may be measured using a continuous method with adouble-wavelength spectrophotometer (e.g., Hemox analyzer, TCS). Thebinding properties of hemoglobin may be assessed using flash photolysis.In this method, the rebinding of CO to intracellular hemoglobintetramers are analyzed at 436 nm after photolysis with a 10 nanosecondpulse at 532 nm.

B. Target Recognition Moieties

The target-binding agents typically include one or more targetrecognition moieties for the selective binding of the composition to atarget molecule. The target recognition moiety is configured tospecifically bind to a target molecule of a particular cell, tissue,receptor, infecting agent or an area of the body of the subject to betreated, such as a target cell, target tissue or target composition.

Examples of target recognition moieties include, but are not limited to,an antigen; ligand; receptor; one member of a specific binding pair;polyamide; peptide; carbohydrate; oligosaccharide; polysaccharide; lowdensity lipoprotein (LDL) or an apoprotein of LDL; steroid; steroidderivative; hormone; hormone-mimic; lectin; drug; antibiotic; aptamer;DNA; RNA; lipid; an antibody; and an antibody-related polypeptide. Inparticular embodiments, the target recognition moiety is an antibody orantibody-related polypeptide. For example, antibodies useful as targetrecognition moieties include antibodies in general and monoclonalantibodies. The target recognition moiety can include a polypeptidehaving an affinity for a polysaccharide target, for example, a lectin(such as a seed, bean, root, bark, seaweed, fungal, bacterial, orinvertebrate lectin). Particularly useful lectins include concanavalinA, which is obtained from jack beans, and lectins obtained from thelentil, Lens culinaris. The target recognition moiety can be a moleculeor a macromolecular structure (e.g., a liposome, a micelle, a lipidvesicle, or the like) that preferentially associates or binds to aparticular tissue, receptor, infecting agent or other area of the bodyof the subject to be treated.

All such targeting methods are contemplated herein for use in theinstant target-binding agents. For non-limiting examples of targetingmethods, See, e.g., U.S. Pat. Nos. 6,316,652; 6,274,552; 6,271,359;6,253,872; 6,139,865; 6,131,570; 6,120,751; 6,071,495; 6,060,082;6,048,736; 6,039,975; 6,004,534; 5,985,307; 5,972,366; 5,900,252;5,840,674; 5,759,542 and 5,709,874.

1. Antibodies as Target Recognition Moieties

Antibodies most ideal for use in subjects are those that arenon-immunogenic when administered to the subject. Such antibodies havethe advantages of exerting minimal side-effects, having long serum andbiologic half-life, having wide bio-distribution, having high targetspecificity and high activity in engaging the effector phase of theimmune system. These antibodies, when intended for human subjects, arecommonly referred to as “humanized,” “human,” “chimeric,” or“primatized” antibodies; these are substantially (>70%) homologous tohuman amino acid sequences.

The target recognition moiety may be an antibody or an antigen bindingantibody fragment configured to specifically bind to at least oneepitope on the target molecule(s) associated with, produced by or on thesurface of a target cell or tissue. The antibody or antibody fragmentmay be monospecific or multispecific. Both polyclonal and monoclonalantibodies may be used, as well as certain recombinant antibodies, suchas chimeric and humanized antibodies and fusion proteins.

The target recognition moiety may be univalent, multivalent and/ormultispecific. By “multivalent” it is meant that the target recognitionmoiety may bind more than one target, which may have the same or adifferent structure, simultaneously. By “multispecific” it is meant thatthe subject agents may bind to at least two targets which are ofdifferent structure. For example, a target recognition moiety having twodifferent specificities would be considered multivalent andmultispecific because it can bind two structurally different targets.

In some instances, the targeting antibody may be part of a multispecificantibody complex with one or more components that bind directly to aspecific protein on the surface of the target cell (See, e.g., U.S. Pat.Nos. 5,470,570 and 5,843,440; U.S. Patent Applications 2003/0215454 A1and 2006/0018912 A1). For example, the targeting antibody may beassociated with a second antibody, such as a red blood cell bindingantibody, that recognizes a protein on the surface of the red bloodcell, e.g., α-N-acetylgalactosaminyltransferase, complement C4,aquaporin, complement decay-accelerating factor, band3 anion transportprotein, Duffy antigen, glycophorin A, B and/or C, galactoside2-L-fucosyltransferase 1, galactoside 2-L-fucosyltransferase 2,galactoside 3(4)-L-fusosyltransferase, CD44, Kell blood groupglycoprotein, urea transporter, complement receptor protein (CR1),membrane transport protein XK, Landsteiner-Wiener blood groupglycoprotein, Lutheran blood group glycoprotein, blood group RH (CE)polypeptide, blood group RH (D) polypeptide, Xg glycoprotein,acetylcholinesterase, anion exchanger, and/or insulin receptor (See,e.g., U.S. Patent Application 2006/0018912 A1). These multispecificantibodies are useful for the assembly of the modified red blood cells.

The antibodies within the multispecific antibody complex may be two ormore intact antibodies and/or two or more antibody fragments such as,for example, Fab′, F(ab′)₂ and/or F, that are linked in some way to oneanother. The two or more antibodies may be fused by chemicalconjugation, crosslinking and/or linker moieties. For example,polypeptides may be covalently bonded to one another through functionalgroups associated with the polypeptides such as, for example, carboxylicacid or free amine groups.

Alternatively, two or more antibodies may be linked through disulfidebonds. For example, the targeting antibody is reacted withN-succinimidyl S-acetylthioacetate (SATA) and subsequently deprotectedby treatment with hydroxylamine to generate an SH-antibody with freesulfhydryl groups (See, e.g., U.S. Patent Application 2003/0215454 A1).The red blood cell binding antibody is reacted with sulfosuccinimidyl4-(N-maelimidomethyl)cyclohexane-1-carboxylate (sSMCC). The twoantibodies treated as such are purified by gel filtration and thenreacted with one another to form a bispecific antibody complex.

Alternatively, the antibodies may be chemically cross-linked to form aheteropolymerized complex using, for example, SPDP[N-succinimidyl-3-(2-pyridyldithio)propionate] (See, e.g., Liu el al.,Proc. Nat'l Acad. Sci. USA 82:8648-8652 (1985); U.S. Pat. No.5,470,570). To generate the complex, the targeting antibody (1-2 mg/ml),for example, is incubated with a 7-fold molar excess of SPDP inphosphate buffered saline (PBS) for 45 minutes at room temperature.Excess SPDP is removed by dialysis overnight against two changes of PBS.Thiol groups are attached to the red blood cell binding antibody, forexample, by incubating the antibody (1-3 mg/ml) with a 1000-fold molarexcess of 2-iminothiolane in 12.5 mM sodium borate/PBS for 45 min atroom temperature. Excess 2-iminothiolane is removed by dialysis asabove. Equimolar amounts of the modified antibodies are incubated for 7h at room temperature and the resulting heteropolymerized complex isseparated from the uncoupled antibodies based on molecular weight usinga standard sizing column.

Fab′ fragments from one or more antibodies may be generated, mixedtogether, and naturally occurring disulfide linkages reformed byoxidation. As such, a subset of the products will contain a Fab′fragment from each antibody. Alternatively, Fab′ fragments from thetargeting antibody, for example, may be activated with a bis-maleimidelinker such as 1,1′-(methylenedi-4,1-phenylene)bis-maleimide and thenlinked to the Fab′ fragments from the red blood cell binding antibodythrough a disulfide bond (See, e.g., U.S. Patent Application2003/0215454 A1).

Alternatively, the two antibody binding activities may be incorporatedinto a single fusion protein using recombinant DNA approaches (See,e.g., U.S. Pat. No. 6,132,992). For example, cDNA encoding the variableregions (V_(L) and V_(H)) of two antibodies directed against separateand distinct antigens, for example, may be combined into a linearexpression construct from which a bispecific single-chain antibody maybe produced (See, e.g., Haisma el al., Cancer Gene Ther. 7:901-904(2000)). As such, cDNA encoding the variable regions (V_(L) and V_(H))of the targeting antibody and of the red blood cell binding antibody,for example, may be manipulated to form a bispecific single-chainantibody.

C. Photoactivatable Molecules

In an embodiment, the target-binding agents may include one or morephotoactivatable molecules, such as a photosensitizer. Typically, thephotoactivatable molecule becomes activated upon exposure toelectromagnetic radiation. Various photoactivatable molecules are usefulover the wavelength range of about 350 to about 1300 nm, the exact rangebeing dependent upon the particular photosensitizer. In suitableembodiments, photoactivatable molecules are those useful in the range ofabout 650-1000 nm (i.e., in the near infrared (“NIR”)). For example,pyropheophorbide and bacteriochloin are useful in about the 650-900 nmrange.

A photoactivatable molecule is a chemical compound that upon exposure tophotoactivating light is activated, releasing a singlet oxygen species.The photoactivatable molecules of the target-binding agents disclosedherein can be any of the variety of synthetic and naturally occurringphotosensitizing agents known in the art, including but not limited to,porphyrins; chlorins; bacteriochlorins; isbacteriochlorins;phthalocyanines; napthalocyanines; porphycenes; porphycyanines;tetra-macrocyclic compounds; poly-macrocyclic compounds;pyropheo-phorbides; pentaphyrin; sapphyrins; texaphyrins; metalcomplexes; tetrahydrochlorins; phonoxazine dyes; phenothiazines;chaloorganapyrylium dyes; rhodamines; fluorescenes; azoporphyrins;benzochlorins; purpurins; chlorophylls; verdins; triarylmethanes;angelicins; chalcogenapyrillium dyes; chlorins; chlorophylls; coumarins;cyanines; ceratin daunomycin; daunomycinone; 5-iminodauno-mycin;doxycycline; furosemide; gilvocarcin M; gilvocarcin V;hydroxy-chloroquine sulfate; lumidoxycycline; mefloquine hydrochloride;mequitazine; merbromin (mercurochrome); primaquine diphosphate;quinacrine dihydrochloride; quinine sulfate; and tetracyclinehydrochloride; certain flavins and related compounds such as alloxazine;flavin mononucleotide; 3-hydroxyflavone; limichrome; limiflavin;6-methylalloxazine; 7-methylalloxazine; 8-methylalloxazine;9-methylalloxazine; 1-methyl limichrome; methyl-2-methoxybenzoate;5-nitrosalicyclic acid; proflavine; and riboflavin; metallo-porphyrins;metallophthalocyanines; methylene blue derivatives; naphthalmides;naphthalocyanines; pheophorbides; pheophytins; photosensitizer dimersand conjugates; phthalocyanines; porphycenes; quinones; retinoids;rhodamines; thiophenes; verdins; vitamins; and xanthene dyes. Generally,any polypyrrolic macrocyclic photosensitive compound that is hydrophobiccan be used.

The release of reactive oxygen species, such as singlet oxygen, maydisrupt active cellular metabolism and cause photodamage by apoptosis.Some photoactivatable molecules, such as phthalocyanines have been shownto cause necrosis by a metabolism-independent mechanism (See, e.g.,Prasad, Introduction to Biophotonics, John Wiley & Sons, Inc. Hoboken,N.J. (2003)). Oxidative degradation of membrane lipids can produce lossof membrane integrity resulting in impairment of membrane transport,rupturing of membrane, increased permeability, andcrosslinking/inactivation of membrane associated polypeptides such asreceptors, enzymes and ion channels. Chlorin, benzoporphyrin, and somephthalocyanine photosensitizers have been shown to cause damage tolysosomes. (See, e.g., Prasad, Introduction to Biophotonics, John Wiley& Sons, Inc. Hoboken, N.J. (2003))

Photoexcitation of the photoactivatable molecules by linear absorption(as opposed to excitation by a nonlinear, two-photon absorption) doesnot require a high peak power or a coherent light source. As such,tungsten and/or mercury or xenon arc lamps may be used to activate thephotoactivatable molecules. Alternatively, lasers may be used for thispurpose. Examples include a dye laser with rhodamine B as lasing mediumand pumped by an argon-ion laser or an intracavity KTP-doubledNd:Vanadate laser, both producing a CW dye laser output in the range of1-4 W. Alternatively, pulse laser sources providing high repetitionrates in the kilohertz range may be used and include gold vapor lasers,copper-pumped dye lasers, and quasi-CW Q-switched Nd:YAG laser-pumpeddye lasers. In some instances, a solid-state diode laser may be usedwith CW and quasi-CW powers in the range of 1-4 W with a single emittersource in the range of 780-850 nm. Other laser sources include tunablesolid-state lasers such a the Ti:sapphire laser (690-1100 nm) and theAlexandrite lasers (720-800 nm) (See, e.g., Prasad, Introduction toBiophotonics, John Wiley & Sons, Inc. Hoboken, N.J. (2003)).

The photoactivatable molecule itself may be monitored by quantitativefluorometry or reflectance spectophotometry. Activation of thephotoactivatable molecules may be assessed by measuring singlet oxygenproduction at about 1270 nm (See, e.g., Lee et al., “Optical Methods forTumor Treatment and Detection: Mechanisms and Techniques in Photodynamictherapy,” XV Biomedical Optics (BiOS) Symposium, San Jose, Calif.(2006)).

A modified red blood cell may be loaded with a photosensitive reagentsuch as, for example, a derivative of hematoporphyrin and subsequentlyirradiated to release a therapeutic agent (See, e.g., Flynn et al.,Cancer Lett. 82:225-229 (1994)). For example, modified red blood cellsare suspended in a physiological buffer such as Ringer's LactateSolution or saline solution with 5% dextrose (w/v) to which is addedhematoporphyrin at a concentration of about 250 μg/ml. The cellsuspension is incubated at 4° C. for 90 min and subsequently washed withthe physiological buffer. The cells may be loaded with a therapeuticagent before, after, or concomitant with hematophorphyrin loading. Themodified red blood cells may be irradiated with a 10 mW output HeNelaser, for example, to induce disruption of modified red blood cells andrelease of the therapeutic agent (See, e.g., Flynn et al., Cancer Lett.82:225-229 (1994)).

Examples of some classes of photoactivatable molecules include, but arenot limited to, angelicins, chalcogenapyrillium dyes, chlorins,chlorophylls, coumarins, cyanines, ceratin daunomycin; daunomycinone;5-iminodauno-mycin; doxycycline; furosemide; gilvocarcin M; gilvocarcinV; hydroxy-chloroquine sulfate; lumidoxycycline; mefloquinehydrochloride; mequitazine; merbromin (mercurochrome); primaquinediphosphate; quinacrine dihydrochloride; quinine sulfate; andtetracycline hydrochloride, certain flavins and related compounds suchas alloxazine; flavin mononucleotide; 3-hydroxyflavone; limichrome;limiflavin; 6-methylalloxazine; 7-methylalloxazine; 8-methylalloxazine;9-methylalloxazine; 1-methyl limichrome; methyl-2-methoxybenzoate;5-nitrosalicyclic acid; proflavine; and riboflavin, metallo-porphyrins,metallophthalocyanines, methylene blue derivatives, naphthalmides,naphthalocyanines, pheophorbides, pheophytins, photosensitizer dimersand conjugates, phthalocyanines, porphycenes, porphyrins, psoralens,purpurins, quinones, retinoids, rhodamines, thiophenes, verdins,vitamins and xanthene dyes (Redmond and Gamlin, Photochem. Photobiol.,70(4):391-475 (1999)).

1. Porphyrins

Examples of porphyrins include 5-azaprotoporphyrin dimethylester;bis-porphyrin; coproporphyrin III; coproporphyrin III tetramethylester;deuteroporphyrin; deuteroporphyrin IX dimethylester;diformyldeutero-porphyrin IX dimethylester; dodecaphenylporphyrin;hematoporphyrin; hematoporphyrin; hematoporphyrin; hematoporphyrin;hematoporphyrin; hematoporphyrin; hematoporphyrin; hematoporphyrin;hematoporphyrin IX; hematoporphyrin monomer; hematoporphyrin dimer;hematoporphyrin derivative; hematoporphyrin derivative; hematoporphyrinderivative; hematoporphyrin derivative A; hematoporphyrin IXdihydrochloride; hematoporphyrin dihydrochloride; hematoporphyrin IXdimethylester; haematoporphyrin IX dimethylester; mesoporphyrindimethylester; mesoporphyrin IX dimethylester;monoformyl-monovinyl-deuteroporphyrin IX dimethylester;monohydroxyethylvinyl deuteroporphyrin;5,10,15,20-tetra(o-hydroxyphenyl)porphyrin;5,10,15,20-tetra(m-hydroxyphenyl)porphyrin;5,10,15,20-tetrakis-(m-hydroxyphenyl)-porphyrin;5,10,15,20-tetra(p-hydroxyphenyl) porphyrin;5,10,15,20-tetrakis(3-methoxyphenyl)-porphyrin;5,10,15,20-tetrakis(3,4-dimethoxyphenyl)porphyrin;5,10,15,20-tetrakis(3,5-dimethoxyphenyl)porphyrin;5,10,15,20-tetrakis(3,4,5-trimethoxyphenyl)porphyrin;2,3,7,8,12,13,17,18-octaethyl-5,10,15,20-tetraphenylporphyrin;Photofring; Photofrin II; porphyrin c; protoporphyrin; protoporphyrinIX; protoporphyrin dimethylester; protoporphyrin IX dimethylester,protoporphyrin propylaminoethylformamide iodide; protoporphyrinN,N-dimethylaminopropyl-formamide; protoporphyrinpropylaminopropylformamide iodide; protoporphyrin butylformamide;protoporphyrin N,N-dimethylamino-formamide; protoporphyrin formamide;sapphyrin 13,12,13,22-tetraethyl-2,7,18,23tetramethylsapphyrin-8,17-dipropanol; sapphyrin23,12,13,22-tetraethyl-2,7,18,23tetramethyl sapphyrin-8-monoglycoside;sapphyrin 3; meso-tetra-(4-N-carboxyphenyl)-porphine;tetra-(3-methoxyphenyl)-porphine;tetra-(3-methoxy-2,4-difluorophenyl)-porphine;5,10,15,20-tetrakis(4-N-methylpyridyl)porphine;meso-tetra-(4-N-methylpyridyl)-porphine tetrachloride;meso-tetra(4-N-methylpyridyl)-porphine;meso-tetra-(3-N-methylpyridyl)-porphine;meso-tetra-(2-N-methylpyridyl)-porphine;tetra(4-N,N,N-trimethylanilinium)porphine;meso-tetra-(4-N,N,N″-trimethylamino-phenyl)porphine tetrachloride;tetranaphthaloporphyrin; 5,10,15,20-tetraphenylporphyrin;tetraphenylporphyrin; meso-tetra-(4-N-sulfonatophenyl)-porphine;tetraphenylporphine tetrasulfonate;meso-tetra(4-sulfonatophenyl)-porphine;tetra(4-sulfonatophenyl)porphine; tetraphenylporphyrin sulfonate;meso-tetra(4-sulfonatophenyl)porphine; tetrakis(4-sulfonatophenyl)porphyrin; meso-tetra(4-sulfonatophenyl)porphine;meso(4-sulfonatophenyl)porphine; meso-tetra(4-sulfonatophenyl)porphine;tetrakis(4-sulfonatophenyl)porphyrin;meso-tetra(4-N-trimethylanilinium)-porphine; uroporphyrin; uroporphyrin1; uroporphyrin IX; and uroporphyrin I.

2. Metalloporphyrins

Examples of metalloporphyrins include cobaltmeso-tetra-(4-N-methylpyridyl)-porphine; cobalt (II)meso(4-sulfonatophenyl)-porphine; copper hermatoporphyrin; coppermeso-tetra-(4-N-methylpyridyl)-porphine; copper (II)meso(4-sulfonatophenyl)-porphine; Europium (III) dimethyltexaphyrindihydroxide; gallium tetraphenylporphyrin; ironmeso-tetra(4-N-methylpyridyl)-porphine; lutetium (III)tetra(N-methyl-3-pyridyl)-porphyrin chloride; magnesium (II)meso-diphenyl tetrabenzoporphyrin; magnesium tetrabenzoporphyrin;magnesium tetraphenylporphyrin; magnesium (II)meso(4-sulfonatophenyl)-porphine; magnesium (II) texaphyrin hydroxidemetalloporphyrin; magnesium meso-tetra-(4-N-methylpyridyl)-porphine;manganese meso-tetra-(4-N-methyl-pyridyl)-porphine; nickelmeso-tetra(4-N-methylpyridyl)-porphine; nickel (II)meso-tetra(4-sulfonatophenyl)-porphine; palladium (II)meso-tetra-(4-N-methylpyridyl)-porphine; palladiummeso-tetra-(4-N-methylpyridyl)-porphine; palladium tetraphenylporphyrin;palladium (II) meso(4-sulfonatophenyl)-porphine; platinum (II)meso(4-sulfonatophenyl)-porphine; samarium (II) dimethyltexaphyrindihydroxide; silver (II) meso(4-sulfonatophenyl)-porphine; tin (IV)protoporphyrin; tin meso-tetra-(4-N-methylpyridyl)-porphine; tinmeso-tetra(4-sulfonatophenyl)-porphine; tin (IV)tetrakis(4-sulfonatophenyl) porphyrin dichloride; cadmium (II)chlorotexaphyrin nitrate; cadmium (II) meso-diphenyltetrabenzoporphyrin; cadmium meso-tetra-(4-N-methylpyridyl)-porphine;cadmium (II) texaphyrin; cadmium (II) texaphyrin nitrate; zinc (11)15-aza-3,7,12,18-tetramethyl-porphyrinato-13,17-diyl-dipropionicacid-dimethylester; zinc (II) chlorotexaphyrin chloride; zinccoproporphyrin III; zinc (II)2,11,20,30-tetra-(1,1-dimethyl-ethyl)tetranaphtho(2,3-b:2′,3′-g:2″3″-1:-2′″3′″-q)porphyrazine;zinc (II)2-(3)-pyridyloxy)benzo[b]-10,19,28-tri(1,1-dimethylethyl)trinaphtho[2′,3′-g:2″3″1::2′″,3′″-q]porphyrazine;zinc (II) 2,18-bis-(3-pyridyloxy)dibenzo[b,1]-10,26-di(1,1-dimethyl-ethyl)dinaphtho-[2′,3′-g:2′″,3′″-q]porphyrazine;zinc (II)2,9-bis-(3-pyridyloxy)dibenzo[b,g]-17,26-di(1,1-dimethyl-ethyl)dinaphtho[-2″,3″-1:2′″,3′″-q]porphyrazine;zinc (II)2,9,16-tris-(3-pyridyloxy)tribenzo[b,g,1]-24=(1,1-dimethyl-ethyl)naphtho[2′″,3′″-q]porphyrazine;zinc (II)2,3-bis-(3-pyridyloxy)benzo[b]-10,19,28-tri(1,1-dimethyl-ethyl)trinaphtho-[2′,3′-g:2″,3″1:2′″,3′″-q]porphyrazine;zinc (II)2,3,18,19-tetrakis-(3-pyridyloxy)dibenzo[b,1]-10,26-di(1,1-dimethyl-ethyl-)trinaphtho[2′,3′-g:2′″,3′″-q]porphyrazine;zinc (II)2,3,9,10-tetrakis-(3-pyridyloxy)dibenzo[b,g]-17,26-di(1,1-dimethyl-ethyl)-dinaphtho[2″,3″-1:2′″,3′″-q]porphyrazine;zinc (II) 2,3,9,10,16,17-hexakis-(3-pyridyloxy)tribenzo[b,g,1]-24(1,1-dimethyl-ethy-l)naphtho[2′″,3′″-q]porphyrazine; zinc (II)2-(3-N-methyl)pyridyloxy)benzo[b]-10,19,28-tri(1,1-dimethyl-ethyl)trinaph-tho[2′,3′-g:2″,3″1:2′″,3′″-q]porphyrazinemonoiodide; zinc (II)2,18-bis-(3-(N-methyl)pyridyloxy)dibenzo[b,1]-10,26-di(1,1-dimethylethyl)-dinaphtho[2′,3′-g:2′″,3′″-q]porphyrazinediiodide; zinc (II)2,9-bis-(3-(N-methyl)pyridyloxy)dibenzo[b,g]-17,26-di(1,1-dimethylethyl)d-inaphtho[2″,3″-1:2′″,3′″-q]porphyrazinediiodide; zinc (II)2,9,16-tris-(3-(N-methyl-pyridyloxy)tribenzo[b,g,1]-24-(1,1-dimethylethyl)naphtho[2′″,3′″-q]porphyrazinetriiodide; zinc (II)2,3-bis-(3-(N-methyl)pyridyloxy)benzo[b]-10,19,28-tri(1,1-dimethylethyl)t-rinaphtho[2′,3′-g:2″,3″-1:2′″,3′″-q]porphyrazinediiodide; zinc (II)2,3,18,19-tetrakis-(3-(N-methyl)pyridyloxy)dibenzo[b,1]-10,26-di(1,1-dimethyl)dinaphtho[2′,3′-g:2′″,3′″-q]porphyrazinetetraiodide; zinc (II)2,3,9,10-tetrakis-(3-(N-methyl)pyridyloxy)dibenzo[g,g]-17,26-di(1,1-dimet-hylethyl)dinaphtho[2″,341-1:2′″,3′″-q]porphyrazinetetraiodide; zinc (II)2,3,9,10,16,17-hexakis-(3-(N-methyl)pyridyloxy)tribenzo[b,g,1]-24-(1-,1-dimethylethyl)naphtho[2′″,3′″-q]porphyrazinehexaiodide; zinc (II) meso-diphenyl tetrabenzoporphyrin; zinc (II)meso-triphenyl tetrabenzoporphyrin; zinc (II)meso-tetrakis(2,6-dichloro-3-sulfonatophenyl)porphyrin; zinc (II)meso-tetra-(4-N-methylpyridyl)-porphine; zinc (II)5,10,15,20-meso-tetra(4-octyl-phenylpropynyl)-porphine; zinc porphyrinc; zinc protoporphyrin; zinc protoporphyrin IX; zinc (II)meso-triphenyl-tetrabenzoporphyrin; zinc tetrabenzoporphyrin; zinc (II)tetrabenzoporphyrin; zinc tetranaphthaloporphyrin; zinctetraphenylporphyrin; zinc (II) 5,10,15,20-tetraphenylporphyrin; zinc(II) meso (4-sulfonatophenyl)-porphine; and zinc (II) texaphyrinchloride.

3. Pheophorbides

Examples of pheophorbides include pheophorbide a; methyl13-1-deoxy-20-formyl-7,8-vic-dihydro-bacterio-meso-pheophorbide a;methyl-2-(1-dodecyloxyethyl)-2-devinyl-pyropheophorbide a;methyl-2-(1-heptyl-oxyethyl)-2-devinyl-pyropheophorbide a;methyl-2-(1-hexyl-oxyethyl)-2-devinyl-pyropheophorbide a;methyl-2-(1-methoxy-ethyl)-2-devinyl-pyropheophorbide a;methyl-2-(1-pentyl-oxyethyl)-2-devinyl-pyropheophorbide a; magnesiummethyl bacteriopheophorbide d; methyl-bacteriopheophorbide d; andpheophorbide.

4. Psoralens

Examples of psoralens include psoralen; 5-methoxypsoralen;8-methoxy-psoralen; 5,8-dimethoxypsoralen; 3-carbethoxypsoralen;3-carbethoxy-pseudopsoralen; 8-hydroxypsoralen; pseudopsoralen;4,5′,8-trimethyl-psoralen; allopsoralen; 3-aceto-allopsoralen;4,7-dimethyl-allopsoralen; 4,7,4′-trimethyl-allopsoralen;4,7,5′-trimethyl-allopsoralen; isopseudopsoralen;3-acetoisopseudopsoralen; 4,5′-dimethyl-isopseudo-psoralen;5′,7-dimethyl-isopseudopsoralen; pseudoisopsoralen;3-aceto-seudoisopsoralen; 3/4′,5′-trimethyl-aza-psoralen;4,4′,8-trimethyl-5′-amino-methylpsoralen;4,4′,8-trimethyl-phthalamyl-psoralen; 4,5′,8-trimethyl-4′-aminomethylpsoralen; 4,5′,8-trimethyl-bromopsoralen; 5-nitro-8-methoxy-psoralen;5′-acetyl4,8-dimethyl-psoralen; 5′-aceto-8-methyl-psoralen; and5′-aceto-4,8-dimethyl-psoralen. Examples of purpurins includeoctaethylpurpurin; octaethylpurpurin zinc; oxidized octaethylpurpurin;reduced octaethylpurpurin; reduced octaethylpurpurin tin; purpurin 18;purpurin-18; purpurin-18-methyl ester; purpurin; tin ethyl etiopurpurinI; Zn(II) aetio-purpurin ethyl ester; and zinc etiopurpurin.

5. Quinones

Examples of quinones include 1-amino-4,5-dimethoxy anthraquinone;1,5-diamino-4,8-dimethoxy anthraquinone; 1,8-diamino-4,5-dimethoxyanthraquinone; 2,5-diamino-1,8-dihydroxy anthraquinone;2,7-diamino-1,8-dihydroxy anthraquinone; 4,5-diamino-1,8-dihydroxyanthraquinone; mono-methylated 4,5- or 2,7-diamino-1,8-dihydroxyanthraquinone; anthralin (keto form); anthralin; anthralin anion;1,8-dihydroxy anthraquinone; 1,8-dihydroxy anthraquinone (Chrysazin);1,2-dihydroxy anthraquinone; 1,2-dihydroxy anthraquinone (Alizarin);1,4-dihydroxy anthraquinone (Quinizarin); 2,6-dihydroxy anthraquinone;2,6-dihydroxy anthraquinone (Anthraflavin); 1-hydroxy anthraquinone(Erythroxy-anthraquinone); 2-hydroxy-anthraquinone;1,2,5,8-tetra-hydroxy anthraquinone (Quinalizarin);3-methyl-1,6,8-trihydroxy anthraquinone (Emodin); anthraquinone;anthraquinone-2-sulfonic acid; benzoquinone; tetramethyl benzoquinone;hydroquinone; chlorohydroquinone; resorcinol; and 4-chlororesorcinol.

6. Retinoids

Examples of retinoids include all-trans retinal; C.sub.17 aldehyde;C.sub.22 aldehyde; 11-cis retinal; 13-cis retinal; retinal; and retinalpalmitate.

7. Rhodamines

Examples of rhodamines include 4,5-dibromo-rhodamine methyl ester;4,5-dibromo-rhodamine n-butyl ester; rhodamine 101 methyl ester;rhodamine 123; rhodamine 6G; rhodamine 6G hexyl ester;tetrabromo-rhodamine 123; and tetramethyl-rhodamine ethyl ester.

8. Other Photoactivatable Molecules

Other non-limiting examples of photoactivatable molecules that may beuseful in the target-binding agents are bacteriochlorophyll-Aderivatives, described in U.S. Pat. Nos. 5,171,741 and 5,173,504;photosensitizing Diels-Alder porphyrin derivatives, described in U.S.Pat. No. 5,308,608; porphyrin-like compounds, described in U.S. Pat.Nos. 5,405,957, 5,512,675, and 5,726,304; imines of porphyrin andporphyrin derivatives, as described in U.S. Pat. Nos. 5,424,305 and5,744,598; alkyl ether analogs of benzoporphyrin derivatives, asdescribed in U.S. Pat. No. 5,498,710; purpurin-18, bacteriopurpurin-18and related compounds, as described in U.S. Pat. No. 5,591,847;meso-substituted chorins, isobacteriochlorins and bacteriochlorins, asdescribed in U.S. Pat. No. 5,648,485; meso-substituted tetramacrocycliccompounds, as described in U.S. Pat. No. 5,703,230; carbodiimide analogsof chlorins and bacteriochlorins, as described in U.S. Pat. No.5,770,730; meso-substituted chlorins, isobacteriochlorins andbacteriochlorins, as described in U.S. Pat. No. 5,831,088; polypyrrolicmacrocycles from meso-substituted tripyrrane compounds, described inU.S. Pat. Nos. 5,703,230, 5,883,246, and 5,919,923; isoimides ofchlorins and bacteriochlorins, described in U.S. Pat. No. 5,864,035;alkyl ether analogs of chlorins having an N-substituted imide ring, asdescribed in U.S. Pat. No. 5,952,366; ethylene glycol esters, describedin U.S. Pat. No. 5,929,105; carotene analogs of porphyrins, chlorins andbacteriochlorins, as described in U.S. Pat. No. 6,103,751; fatty acidester derivatives of porphyrin, chlorin, or bacteriochlorin, asdescribed in U.S. Pat. No. 6,245,811; indium photosensitizers, asdescribed in U.S. Pat. No. 6,444,194; porphyrins, chlorins,bacteriochlorins, and related tetrapyrrolic compounds described in U.S.Pat. No. 6,534,040; 1,3-propane diol ester and ether derivatives ofporphyrins, chlorins and bacteriochlorins, as described in U.S. Pat. No.6,555,700; trans beta substituted chlorins, as described in U.S. Pat.No. 6,559,374; and palladium-substituted bacteriochlorophyl derivatives,as described in U.S. Pat. No. 6,569,846; and the photosensitizerentities disclosed in Wilson et al., (Curr. Micro. 25:77-81 (1992)) andin Okamoto et al., (Lasers in Surg. Med. 12:450-485 (1992)). Generallyany hydrophobic or hydrophilic photosensitizing agent, that absorbs inthe ultra-violet, visible and infra-red spectroscopic ranges, would beuseful in the disclosed conjugates.

D. Quencher Molecules

In various embodiments, the target-binding agents include a quenchermolecule. In an embodiment, a light quencher is provided to preventactivation of the photoactivatable molecule if the targeting compositionis not bound to a target molecule. Alternatively, the quencher maycapture singlet oxygen from the photoactivatable molecule in situationswhere the target-binding agent is not bound to the target.

In an embodiment, the quencher molecule quenches the excited state ofthe photoactivatable molecule. For example, upon binding of thetarget-binding agent to its target, the three dimensional structure ofthe target-binding agent is altered in such a way that the quenchingagent is no longer positioned close enough to quench the excited stateof the photoactivatable molecule, thus allowing the photoactivatablemolecule to function as required for generation of singlet oxygen. Thesinglet oxygen is then available to destroy the target or lyse themodified red blood cell. The quenching agent serves to prevent thegeneration of false positive signals from the photoactivatable moleculewhen it is not bound to the target.

In a specific embodiment, the photoactivatable molecule is a porphyrinor porphyrin derivative tetrapyrrole that includes a metal atom in itscentral coordination cavity and the quencher comprises one or moresuitable functional groups that coordinate to the axial position of themetal coordinated within the photoactivatable molecule. The targetrecognition moiety is positioned in the agent in such a way that theinteraction of the target recognition moiety with the target disruptsthe association of the axial ligand to the metal, releasing thequenching agent and allowing the porphyrin or porphyrin derivativetetrapyrrole to be activated when irradiated.

In an embodiment, the quencher molecule is a light quencher, whichprevents light of a suitable wavelength from exciting the photoactivablemolecule. For instance, the quencher may absorb photons of a particularwavelength before those photons activate the photoactivatable molecule.Suitable light quenchers may include4-(4′-dimethylamino-phenylazo)benzoic acid (Dabcyl) or dark quenchers,such as black hole quenchers sold under the tradename “BHQ” (e.g.,BHQ-0, BHQ-1, BHQ-2, and BHQ-3, Biosearch Technologies, Novato, Calif.).Dark quenchers also may include quenchers sold under the tradename“QXLTM” (Anaspec, San Jose, Calif.). Dark quenchers also may includeDNP-type non-fluorophores that include a 2,4-dinitrophenyl group.

In an embodiment, the quencher molecule is an antioxidant which capturessinglet oxygen produced by the photoactivatable molecule before it cancause damage to surround cells or tissues. Suitable quenchers forsinglet oxygen include, but are not limited to, glutathione, trolox,flavonoids, vitamin C, vitamin E, cysteine and ergothioneine and othernon-toxic quenchers.

E. Molecules

In an embodiment, the red blood cells may be modified with fusionmolecules or fusogens known to facilitate fusion with other cells. Uponfusion, the modified red blood cell may release its loaded content suchas, for example, an anti-cancer therapeutic agent or a photosensitivereagent. For instance, breast cancer cells have been shown to express anendogenous retroviral envelope protein, syncytin-1, that enables thetumor cells to fuse in vivo with endothelial cells expressing acorresponding D-type retroviral receptor, the Na+-dependent neutralamino acid transporter ASCT2 (See, e.g, Larsson el al., Scientific WorldJournal 7:1193-1197 (2007)). Syncytin-1 is also expressed by endometrialcarcinomas. As such, red blood cells may be modified with a syncytin-1interacting receptor such as, for example, ASCT2 that would enable themodified red blood cells to fuse with cancer cells. For example, cDNAencoding human ASCT2 may be cloned using sequence information availablein NCBI/GenBank (See, e.g., accession number NP_(—)005619).Alternatively, cDNA encoding human ASCT2 may be acquired from acommercial source (e.g., OriGene Technologies, Inc., Rockville, Md.,USA). The cDNA is cloned into an appropriate expression vector andsubsequently transfected into cultured hematopoietic stem cells.Alternatively, the cDNA encoding ASCT2 may be transcribed to generatemRNA which is subsequently introduced into isolated reticulocytes asdescribed above.

II. Assembly of the Target-Binding Agents

A. Attachment of a Target Recognition Moiety to a PhotoactivatableMolecule and a Quencher Molecule

In an embodiment, the target recognition moiety of the target-bindingagent is conjugated to a photoactivable molecule and a quenchermolecule. Upon binding of the target recognition moiety to the targetmolecule, the quencher molecule is released or otherwise separated fromthe photoactivateable molecule. In the “unquenched” state, thephotoactivatable molecule may be activated by light of a suitablewavelength. The conjugation of these molecules is typically by way ofattachment sites. Most attachments are conveniently effected viasulfhydryl or amine interactions. Synthetic and commercial alternativesare available depending on the selected photoactivable molecule, orquencher molecule. The distance between the photoactivatable moleculeand the quencher molecule is selected so that interaction of the targetrecognition moiety results in repositioning of the quencher molecule. Ifthe photoactivatable molecule and the quencher are too close, theninteraction of the target recognition moiety with the target may not endquenching of photoactivatable molecule. If the distance between thephotoactivatable molecule and the quencher molecule is too great, thenthe quencher molecule may not prevent all electromagnetic radiation fromreaching the photoactivatable molecule. The distances can be determinedby any method, such as by calculation or empirically.

Techniques in synthetic chemistry provide methods for the attachment ofphotoactivatable molecule and/or quencher molecule to the targetrecognition moiety. For example, synthetic linkage techniques are knownthat allow incorporation of both various types of molecules, including aphotoactivatable molecule and an quencher molecule within anoligonucleotide (See U.S. Pat. No. 4,996,143). There is extensiveguidance in the literature for derivatizing photoactivatable andquencher molecules for covalent attachment via readily availablereactive groups that can be added to a molecule. The diversity andutility of chemistries available for conjugating molecules and surfacesis exemplified by the extensive body of literature on preparing nucleicacids derivatized with fluorophores. See, for example, Ullhman et al.,U.S. Pat. No. 3,996,345 and Khanna et al., U.S. Pat. No. 4,351,760.

The target-binding agents disclosed herein can be conjugated by using acoupling agent. Any bond which is capable of linking the components suchthat they are stable under physiological conditions for the time neededfor administration and treatment is suitable, but covalent linkages arepreferred. The link between two components may be direct, e.g., where aphotoactivatable molecule is linked directly to a target recognitionmoiety, or indirect, e.g., where a photoactivatable molecule is linkedto a linking component and that linking component being linked to thetarget recognition moiety.

A coupling agent should function under conditions of temperature, pH,salt, solvent system, and other reactants that substantially retain thechemical stability of the photoactivatable molecule, the quenchermolecule and the target recognition moiety. Coupling agents should linkthe component moieties stably, but such that there is only minimal or nodenaturation or deactivation of the photoactivatable molecule, quenchermolecule or the target recognition moiety. Many coupling agents reactwith an amine and a carboxylate, to form an amide, or an alcohol and acarboxylate to form an ester. Coupling agents are known in the art (See,e.g., Bodansky, Principles of Peptide Synthesis, 2nd ed, John Wiley,N.Y. (1991), and Greene & Wuts, Protective Groups in Organic Synthesis,2nd ed, John Wiley, N.Y. (1991)). Representative combinations of suchgroups are amino with carboxyl to form amide linkages, or carboxy withhydroxy to form ester linkages or amino with alkyl halides to formalkylamine linkages, or thiols with thiols to form disulfides, or thiolswith maleimides or alkyl halides to form thioethers. Obviously,hydroxyl, carboxyl, amino and other functionalities, where not presentmay be introduced by known methods.

The target-binding agents provided herein can be prepared by couplingthe photoactivatable molecule to a target recognition moiety, such as anantibody, by cleaving an available ester moiety on the photoactivatablemolecule and coupling the compound via peptide linkages to an antibodythrough an N terminus, or by other methods known in the art. A varietyof coupling agents, including cross-linking agents, can be used forcovalent conjugation. Examples of cross-linking agents includeN,N′-dicyclohexylcarbodiimide (DCC), N-succinimidyl-S-acetyl-thioacetate(SATA), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP),ortho-phenylene-dimaleimide (o-PDM), and sulfosuccinimidyl4N-maleimido-methyl)-cyclohexane-1-carboxylate (sulfo-SMCC). See, e.g.,Karpovsky et al., J. Exp. Med. 160:1686 (1984); and Liu M A et al.,Proc. Natl. Acad. Sci. USA 82: 8648 (1985). Other methods include thosedescribed by Brennan et al., Science 229: 81-83 (1985) and Glennie etal., J. Immunol. 139: 2367-2375 (1987). A large number of couplingagents for peptides and proteins, along with buffers, solvents, andmethods of use, are described in the Pierce Chemical Co. catalog, pagesO-90 to O-110 (1995, Pierce Chemical Co., 3747 N. Meridian Rd., RockfordIll., 61105, U.S.A.).

For example, DCC is a useful coupling agent that can be used to promotecoupling of the alcohol NHS to chlorin e6 in DMSO forming an activatedester which can be cross-linked to polylysine. DCC is a carboxy-reactivecross-linker commonly used as a coupling agent in peptide synthesis.Another useful cross-linking agent is SPDP, a heterobifunctionalcross-linker for use with primary amines and sulfhydryl groups. SPDP hasa molecular weight of 312.4, a spacer arm length of 6.8 angstroms, isreactive to NHS-esters and pyridyldithio groups, and produces cleavablecross-linking such that, upon further reaction, the agent is eliminatedso the photoactivatable molecule can be linked directly to a linkingcomponent or target recognition moiety. Other useful conjugating agentsare SATA for introduction of blocked SH groups for two-stepcross-linking, which is deblocked with hydroxylamine-HCl, andsulfo-SMCC, reactive towards amines and sulfhydryls. Other cross-linkingand coupling agents are also available from Pierce Chemical Co.Additional compounds and processes, particularly those involving aSchiff base as an intermediate, for conjugation of proteins to otherproteins or to other compositions, for example to reporter groups or tochelators for metal ion labeling of a protein, are disclosed in EPO243,929 A2 (published Nov. 4, 1987).

Reactive Groups. The photoactivatable molecule or target recognitionmoiety can be conjugated, directly or through a linking component, tothe quencher molecule using reactive groups, either on the donormolecule or on the acceptor molecule or the targeting moiety. Forexample, molecules that contain carboxyl groups can be joined tolysine-amino groups in the target polypeptides either by preformedreactive esters (such as N-hydroxy succinimide ester) or estersconjugated in situ by a carbodiimide-mediated reaction. The same appliesto molecules that contain sulfonic acid groups, which can be transformedto sulfonyl chlorides which react with amino groups. Molecules that havecarboxyl groups can be joined to amino groups, such as on a polypeptide,by an in situ carbodiimide method. Molecules can also be attached tohydroxyl groups of serine or threonine residues or to sulfhydryl groupsof cysteine residues.

Methods of joining components of a target-binding agent can useheterobifunctional cross linking reagents. These agents bind afunctional group in one chain and to a different functional group in thesecond chain. These functional groups typically are amino, carboxyl,sulfhydryl, and aldehyde. There are many permutations of appropriatemoieties which will react with these groups and with differentlyformulated structures, to conjugate them together. (See Merrifield etal., Ciba Found Symp. 186: 5-20 (1994)).

The photoactivatable molecule of the target-binding agent may beoptionally functionalized so as to include a linking component whichallows the photoactivatable molecule to be linked to a targetrecognition moiety, such as an analyte, antigen, antibody or othermolecule. For example, the linking component may include, but is notlimited to, an oligonucleotide, a polynucleotide, a nucleic acid, anoligosaccharide, a polysaccharide or a diaminoalkane linking species,such as 1,3-diaminopropane. A variety of linking components which aresuited to this purpose have been described. For example, see Kricka,Ligand-Binder Assays, Labels and Analytical Strategies, pp. 15-51,Marcel Dekker, Inc., New York, N.Y. (1985)). The photoactivatablemolecule is linked to the linking component and the linking component islinked to the analyte, antigen, antibody or other molecule usingconventional techniques.

Reactive Groups and Reactions. Reactive groups and classes of reactionsuseful in preparing the disclosed conjugates are generally those thatare well known in the art of bioconjugate chemistry. Classes ofreactions include those that proceed under relatively mild conditions.These include, but are not limited to nucleophilic substitutions (e.g.,reactions of amines and alcohols with acyl halides, active esters),electrophilic substitutions (e.g., enamine reactions) and additions tocarbon-carbon and carbon-heteroatom multiple bonds (e.g., Michaelreaction). These and other useful reactions are discussed in, forexample, Morrison et al., Organic Chemistry, 4th Ed., Allyn and Bacon,Inc. (1983), and Hermanson, Bioconjugate Techniques, Academic Press, SanDiego(1996).

For example, useful reactive functional groups include: (a) carboxylgroups and various derivatives thereof including, but not limited to,N-hydroxysuccinimide esters, N-hydroxybenztriazole esters, acid halides,acyl imidazoles, thioesters, p-nitrophenyl esters, alkyl, alkenyl,alkynyl and aromatic esters; (b) hydroxyl groups, which can be convertedto esters, ethers, aldehydes, etc.; (c) haloalkyl groups, wherein thehalide can be later displaced with a nucleophilic group such as, forexample, an amine, a carboxylate anion, thiol anion, carbanion, or analkoxide ion, thereby resulting in the covalent attachment of a newgroup at the site of the halogen atom; (d) dienophile groups, which arecapable of participating in Diels-Alder reactions such as, for example,maleimido groups; (e) carbonyl groups, such that subsequentderivatization is possible via formation of carbonyl derivatives suchas, for example, imines, hydrazones, semicarbazones or oximes, or viasuch mechanisms as Grignard addition or alkyllithium addition; (f)sulfonyl groups for subsequent reaction with amines, for example, toform sulfonamides; (g) thiol groups, which can be converted todisulfides or reacted with acyl halides; (h) amine or sulfhydryl groups,which can be, for example, acylated, alkylated or oxidized; (i) alkenes,which can undergo, for example, cycloadditions, acylation, Michaeladdition, etc; (j) epoxides, which can react with, for example, aminesand hydroxyl compounds; and (k) phosphoramidites and other standardfunctional groups useful in nucleic acid synthesis.

B. Placement of the Photoactivatable Molecule and Quencher Molecule

The photoactivatable molecule and quencher molecule of thetarget-binding agents disclosed herein are positioned to be in aconfiguration so that the agent is in a “quenched state” when it is notinteracting with a target molecule. When the agent interacts with atarget via the target recognition moiety, the photoactivatable moleculeand the quencher molecule are separated. Thus, the spatial rearrangementof the photoactivatable molecule and quencher in the target-bindingagent occurs only after interaction of the target recognition moietywith its target. Hence, the target recognition moiety is selected andpositioned in the conjugate so that when the target recognition moietyinteracts with its target, the spatial arrangement of the agent ischanged such that the photoactivatable molecule is are no longer in aquenched state.

C. Conjugation of Target-binding Agents and/or Fusion Molecules to RedBlood Cells

In an embodiment, the target-binding agent may be bound to the surfaceof a modified red blood cell through a biotin-streptavidin bridge. Forexample, a biotinylated antibody may be linked to a non-specificallybiotinylated cell surface through a streptavidin bridge. In anembodiment, the target-binding agent is attached to the red blood cellvia the target recognition moiety, e.g., antibody. Antibodies can beconjugated to biotin by a number of chemical means (See, e.g, Hirsch etal., Methods Mol. Biol. 295: 135-154 (2004)). The surface membraneproteins of a red blood cell may be biotinylated using an amine reactivebiotinylation reagent such as, for example, EZ-Link Sulfo-NHS-SS-Biotin(sulfosuccinimidyl 2-(biotinamido)-ethyl-1,3-dithiopropionate;Pierce-Thermo Scientific, Rockford, Ill., USA; See, e.g., Jaiswal etal., Nature Biotech. 21:47-51 (2003)). Isolated red blood cells may beincubated for 30 min at 4° C. in 1 mg/ml solution of sulfo-NHS-SS inphosphate-buffered saline. Excess biotin reagent is removed by washingthe cells with Tris-buffered saline, for example. The biotinylated cellsare then reacted with the biotinylated antibody in the presence ofstreptavidin to form the modified red blood cells.

In another embodiment, the target-binding agent may be attached to thesurface of the modified red blood with a bispecific antibody, forexample, with both target cell and red blood cell binding activities.The number of antigen binding sites on the modified red blood cell mayrange from about 0 to over 1000 sites, for example, depending upon thebinding conditions (See, e.g., U.S. Pat. No. 5,470,570). The red bloodcells may be further modified as described herein and re-introduced intoan individual.

Alternatively, the bispecific antibody, for example, may be addeddirectly to the bloodstream where it optimally binds in vivo to themodified red blood cell and to the target cell (See, e.g., U.S. PatentApplication 2003/0215454). Alternatively, a unique receptor molecule maybe expressed on the surface of a modified red blood cell that isdetected by the bispecific antibody to ensure the selectivity ofbispecific antibody to the modified red blood cell.

For example, the following receptors can be used to target macrophages:the complement receptor (Rieu et al., J. Cell Biol. 127:2081-2091(1994)), the scavenger receptor (Brasseur et al., Photochem. Photobiol.69:345-352 (1999)), the transferrin receptor (Dreier et al., Bioconjug.Chem. 9:482-489 (1998); Hamblin et al., J. Photochem. Photobiol. 26:4556(1994)); the Fc receptor (Rojanasakul et al., Pharm. Res. 11:1731-1733(1994)); the mannose receptor (Frankel et al., Carbohydr. Res.300:251-258 (1997); Chakrabarty et al., J. Protozool. 37:358-364(1990)). Target recognition moieties that can be conjugated withphotoactivatable molecules, for example to target to macrophages,include low density lipoproteins (Mankertz et al., Biochem. Biophys.Res. Commun. 240:112-115 (1997); von Baeyer et al., Int. J. Clin.Pharmacol. Ther. Toxicol. 31:382-386 (1993)), very low densitylipoproteins (Tabas et al., J. Cell Biol. 115:1547-1560 (1991)), mannoseresidues and other carbohydrate moieties (Pittet et al., Nucl. Med.Biol. 22:355-365 (1995)), poly-cationic molecules, such as poly-L-lysine(Hamblin et al., J. Photochem. Photobiol. 26:45-56 (1994)), liposomes(Bakker-Woudenberg et al., J. Drug Target. 2:363-371 (1994); Betageri etal., J. Pharm. Pharmacol. 45:48-53 (1993)), antibodies (Gruenheid etal., J. Exp. Med. 185:717-730, (1997)), and 2-macroglobulin (Chu et al.,J. Immunol. 152:1538-1545 (1994)).

In another embodiment, the target-binding agent is attached to the redblood cell via a covalent attachment. For example, the targetrecognition moiety may be derivatized and bound to the red blood cellusing a coupling compound containing an electrophilic group that willreact with nucleophiles on the red blood cell to form the interbondedrelationship. Representative of these electrophilic groups are α,βunsaturated carbonyls, alkyl halides and thiol reagents such assubstituted maleimides. In addition, the coupling compound can becoupled to the target recognition moiety via one or more of thefunctional groups in the target recognition moiety such as amino,carboxyl and tryosine groups. For this purpose, coupling compoundsshould contain free carboxyl groups, free amino groups, aromatic aminogroups, and other groups capable of reaction with enzyme functionalgroups. Highly charged derivatives of target recognition moiety can alsobe prepared for immobilization on erythrocytes through electrostaticbonding. Examples of these derivatives would include polylysyl andpolyglutamyl enzymes.

The choice of the reactive group embodied in the derivative depends onthe reactive conditions employed to couple the electrophile with thenucleophilic groups on the red blood cell for immobilization. Acontrolling factor is the desire not to inactivate the coupling agentprior to coupling of the target recognition moiety immobilized by theattachment to the red blood cell.

Such coupling immobilization reactions can proceed in a number of ways.Typically, a coupling agent can be used to form a bridge between themacromolecule and the red blood cell. In this case, the coupling agentshould possess a functional group such as a carboxyl group which can becaused to react with the target recognition moiety. One pathway forpreparing the macromolecular derivative comprises the utilization ofcarboxyl groups in the coupling agent to form mixed anhydrides whichreact with the target recognition moiety, in which use is made of anactivator which is capable of forming the mixed anhydride.Representative of such activators are isobutylchloroformate or otherchloroformates which give a mixed anhydride with coupling agents such as5,5′-(dithiobis (2-nitrobenzoic acid) (DTNB), p-chloromercuribenzoate(CMB), or m-maleimidobenzoic acid (MBA). The mixed anhydride of thecoupling agent reacts with the target recognition moiety to yield thereactive derivative which in turn can react with nucleophilic groups onthe red blood cell to immobilize the macromolecule.

Functional groups on the target recognition moiety such as carboxylgroups can be activated with carbodiimides and the like activators.Subsequently, functional groups on the bridging reagent, such as aminogroups, will react with the activated group on the target recognitionmoiety to form the reactive derivative. In addition, the coupling agentshould possess a second reactive grouping which will react withappropriate nucleophilic groups on the red blood cell to form thebridge. Typical of such reactive groupings are alkylating agents such asiodoacetic acid, α, β unsaturated carbonyl compounds, such as acrylicacid and the like, thiol reagents, such as mercurials, substitutedmaleimides and the like.

Alternatively, functional groups on the target recognition moiety can beactivated so as to react directly with nucleophiles on red blood cellsto obviate the need for a bridge-forming compound. For this purpose,beneficial use is made of an activator such as Woodward's Reagent K orthe like reagent which brings about the formation of carboxyl groups inthe target recognition moiety into enol esters, as distinguished frommixed anhydrides. The enol ester derivatives of target recognitionmoieties will subsequently react with nucleophilic groups on the redblood cell to effect immobilization of the macromolecule.

D. Genetically Engineered Red Blood Cells

In an embodiment, red blood cell precursor cells are geneticallyengineered to express one or more protein- or RNA-based pharmaceuticalsand/or one or more imaging agents (e.g., a fluorescent protein). Thissection describes the transformation of reticulocytes and hematopoieticstem cells, which are both precursor cells for mature erythrocytes.

1. Transformation of Reticulocytes

Isolated reticulocytes may be transfected with mRNA encoding proteinsand/or peptides of interest. Messenger RNA may be derived from in vitrotranscription of a cDNA plasmid construct containing the coding sequencecorresponding to the protein and/or peptide of interest. For example,the cDNA sequence corresponding to the protein and/or peptide ofinterest may be inserted into a cloning vector containing promotersequence compatible with specific RNA polymerases. For example, thecloning vector ZAP Express® pBK-CMV (Stratagene, La Jolla, Calif., USA)contains T3 and T7 promoter sequence compatible with T3 and T7 RNApolymerase, respectively. For in vitro transcription of sense mRNA, theplasmid is linearized at a restriction site downstream of the stopcodon(s) corresponding to the end of the coding sequence of the proteinand/or peptide of interest. The mRNA is transcribed from the linear DNAtemplate using a commercially available kit such as, for example, theRNAMaxx(& High Yield Transcription Kit (from Stratagene, La Jolla,Calif., USA). In some instances, it may be desirable to generate5′-m⁷GpppG-capped mRNA. As such, transcription of a linearized cDNAtemplate may be carried out using, for example, the mMESSAGE mMACHINEHigh Yield Capped RNA Transcription Kit from Ambion (Austin, Tex., USA).Transcription may be carried out in a reaction volume of 20-100 μl at37° C. for 30 min to 4 h. The transcribed mRNA is purified from thereaction mix by a brief treatment with DNase I to eliminate thelinearized DNA template followed by precipitation in 70% ethanol in thepresence of lithium chloride, sodium acetate or ammonium acetate. Theintegrity of the transcribed mRNA may be assessed using electrophoresiswith an agarose-formaldehyde gel or commercially available Novexpre-cast TBE gels (e.g., Novex, Invitrogen, Carlsbad, Calif., USA).

Messenger RNA encoding proteins and/or peptides of interest may beintroduced into reticulocytes using a variety of approaches including,for example, lipofection and electroporation (van Tandeloo et al., Blood98:49-56 (2001)). For lipofection, for example, 5 μg of in vitrotranscribed mRNA in Opti-MEM (Invitrogen, Carlsbad, Calif., USA) isincubated for 5-15 min at a 1:4 ratio with the cationic lipid DMRIE-C(Invitrogen). Alternatively, a variety of other cationic lipids orcationic polymers may be used to transfect cells with mRNA including,for example, DOTAP, various forms of polyethylenimine, and polyL-lysine(Sigma-Aldrich, Saint Louis, Mo., USA), and Superfect (Qiagen, Inc.,Valencia, Calif., USA; See, e.g., Bettinger et al., Nucleic Acids Res.29:3882-3891 (2001)). The resulting mRNA/lipid complexes are incubatedwith cells (1-2×10⁶ cells/ml) for 2 h at 37° C., washed and returned toculture. For electroporation, for example, about 5 to 20×10⁶ cells in500 μl of Opti-MEM (Invitrogen, Carlsbad, Calif., USA) are mixed withabout 20 μg of in vitro transcribed mRNA and electroporated in a 0.4-cmcuvette using, for example, and Easyject Plus device (EquiBio, Kent,United Kingdom). In some instances, it may be necessary to test variousvoltages, capacitances and electroporation volumes to determine theoptimal conditions for transfection of a particular mRNA into areticulocyte. In general, the electroporation parameters required toefficiently transfect cells with mRNA appear to be less detrimental tocells than those required for electroporation of DNA (van Tandeloo etal., Blood 98:49-56 (2001)).

Alternatively, mRNA may be transfected into a reticulocyte using apeptide-mediated RNA delivery strategy (See, e.g., Bettinger et al.,Nucleic Acids Res. 29:3882-3891 (2001)). For example, the cationic lipidpolyethylenimine 2 kDA (Sigma-Aldrich, Saint Louis, Mo., USA) may becombined with the melittin peptide (Alta Biosciences, Birmingham, UK) toincrease the efficiency of mRNA transfection, particularly inpost-mitotic primary cells. The mellitin peptide may be conjugated tothe PEI using a disulfide cross-linker such as, for example, thehetero-bifunctional cross-linker succinimidyl 3-(2-pyridyldithio)propionate. In vitro transcribed mRNA is preincubated for 5 to 15 minwith the mellitin-PEI to form an RNA/peptide/lipid complex. This complexis then added to cells in serum-free culture medium for 2 to 4 h at 37°C. in a 5% CO₂ humidified environment and then removed and thetransfected cells allowed to continue growing in culture.

2. Transformation of Hematopoetic Stem Cells

Non-endogenous proteins such as, for example, receptors, enzymes and/ortherapeutic peptides may be genetically introduced into hematopoieticstem cells prior to terminal differentiation using a variety of DNAtechniques, including transient or stable transfections and gene therapyapproaches. These non-endogenous proteins expressed on the surfaceand/or in the cytoplasm of mature red blood cell may be used to targetthe modified red blood cell to a specific location, to bind specificblood analytes, to react and/or signal in the presence of specificanalytes, and/or to treat a specific disease or condition.

Viral based gene transfer. Viral gene transfer may be used to transfecthematopoietic stem cells with DNA encoding proteins and/or peptides ofinterest (Papapetrou et al., Gene Therapy 12:S118-S130 (2005)). A numberof viruses may be used as gene transfer vehicles including Moloneymurine leukemia virus (MMLV), adenovirus, adeno-associated virus, herpessimplex virus (HSV), lentiviruses such as human immunodeficiency virus 1(HIV 1), and spumaviruses such as foamy viruses, for example (See, e.g.,Osten et al., HEP 178:177-202 (2007)). Retroviruses, for example,efficiently transduce mammalian cells including human cells andintegrate into chromosomes, conferring stable gene transfer.

A cell membrane associated receptor, for example, may be transcribedinto hematopoietic stem cells and subsequently expressed in a mature redblood cell using a Moloney murine leukemia virus (MMLV) vector backbone(Malik et al., Blood 91:2664-2671 (1998)). Vectors based on MMLV, anoncogenic retrovirus, are currently used in gene therapy clinical trials(Hossle et al., News Physiol. Sci. 17:87-92 (2002)). A DNA constructcontaining the cDNA encoding a cell membrane associated receptor suchas, for example, the mu opioid receptor is generated in the MMLV vectorbackbone using standard molecular biology techniques. The construct istransfected into a packaging cell line such as, for example, PA317 cellsand the viral supernatant is used to transfect producer cells such as,for example, PG13 cells. The PG13 viral supernatant is incubated withhematopoietic stem cells that have been isolated and cultured asdescribed in above. The expression of the cell membrane associatedreceptor such as, for example, the mu opioid receptor may be monitoredusing FACS analysis (fluorescence-activated cell sorting), for example,with a fluorescently labeled antibody directed against the cell membraneassociated receptor. Similar methods may be used to express acytoplasmic protein such as, for example, a modified hemoglobin molecule(See, e.g., Nicolini et al., Blood 100: 1257-1264 (2002)) or a smallpeptide such as, for example, a cytokine (See, e.g., Song et al., CancerRes. 66:6304-6311 (2006)) in a hematopoietic stem cell.

Similarly, a fluorescent tracking molecule such as, for example, greenfluorescent protein (GFP) may be transfected into hematopoietic stemcells using a viral-based approach (Tao et al., Stem Cells 25:670-678(2007)). As such, bone marrow cells are isolated and cultured asdescribed herein. Two days prior to transfection, the cells areprestimulated in minimum essential medium (MEM) containing 20% fetalbovine serum, 4 mM L-glutamine, 100 units/ml penicillin, 100 μg/mlstreptomycin, 100 ng/ml murine stem cell factor, 100 ng/ml murineFLT3-ligand, and 100 ng/ml murine thrombopoietin. Ecotopic retroviralvectors containing DNA encoding the enhanced green fluorescent protein(EGFP) or a red fluorescent protein (e.g., DsRed-Express) are packagedusing a packaging cell such as, for example, the Phoenix-Eco cell line(distributed by Orbigen, San Diego, Calif.). Packaging cell lines stablyexpress viral proteins needed for proper viral packaging including, forexample, gag, pol, and env. Supernatants from the Phoenix-Eco cells intowhich viral particles have been shed are used to transduce prestimulatedhematopoietic stem cells. In some instances, transduction may beperformed on a specially coated surface such as, for example, fragmentsof recombinant fibronectin to improve the efficiency of retroviralmediated gene transfer (e.g., RetroNectin, Takara Bio USA, Madison,Wis.). As such, prestimulated cells are incubated in RetroNectin-coatedplates with retroviral Phoenix-Eco supernatants plus 100 ng/ml murinestem cell factor, 100 ng/ml murine FLT3-ligand, and 100 ng/ml murinethrombopoietin. After incubation at 37° C., plates are centrifuged at400×g for 5 min at 20° C. and further incubated at 37° C. for 5.5 h.Transduction may be repeated the next day. In this instance, thepercentage of cells expressing EGFP or DsRed-Express may be assessed byFACS. Other reporter genes that may be used to assess transductionefficiency include, for example, beta-galactosidase, chloramphenicolacetyltransferase, and luciferase as well as low-affinity nerve growthfactor receptor (LNGFR), and the human cell surface CD24 antigen(Bierhuizen et al., Leukemia 13:605-613 (1999)).

Non-viral gene transfer. Nonviral vectors may be used to introducegenetic material into hematopoietic stem cells (Papapetrou et al., GeneTherapy 12:S118-S130 (2005)). Nonviral-mediated gene transfer differsfrom viral-mediated gene transfer in that the plasmid vectors contain noproteins, are less toxic and easier to scale up, and have no host cellpreferences. The “naked DNA” of plasmid vectors are by themselvesinefficient in delivering genetic material to a cell and therefore arecombined with a gene delivery method that enables entry into cells. Anumber of delivery methods may be used to transfer nonviral vectors intohematopoietic stem cells including chemical and physical methods.

A nonviral vector encoding a protein and/or peptide of interest may beintroduced into hematopoietic stem cells using synthetic macromoleculessuch as cationic lipids and polymers (Papapetrou et al., Gene Therapy12:S118-S130 (2005)). Cationic liposomes, for example form complexeswith DNA through charge interactions. The positively charged DNA/lipidcomplexes bind to the negative cell surface and are taken up by the cellby endocytosis. This approach may be used, for example, to transfecthematopoietic cells (See, e.g., Keller et al., Gene Therapy 6:931-938(1999)). Hematopoietic cells are cultured in association with adherentstromal cells as described herein. The plasmid DNA (approximately 0.5 μgin 25-100 μL of a serum free medium, such as, for example, OptiMEM(Invitrogen, Carlsbad, Calif.)) is mixed with a cationic liposome(approximately 4 μg in 25 μL of serum free medium) such as thecommercially available transfection reagent Lipofectamine™ (Invitrogen,Carlsbad, Calif.) and allowed to incubate for at least 20 min to formcomplexes. The DNA/liposome complex is added to the hematopoietic cellsand allowed to incubate for 5-24 h, after which time transgeneexpression may be assayed. Alternatively, other commercially availableliposome tranfection agents may be used (e.g., In vivo GeneSHUTTLE™,Qbiogene, Carlsbad, Calif.).

Alternatively, a cationic polymer such as, for example, polyethylenimine(PEI) may be used to efficiently transfect hematopoietic and umbilicalcord blood-derived CD34+ cells (See, e.g., Shin et al., Biochim.Biophys. Acta 1725:377-384 (2005)). Human CD34+ cells are isolated fromhuman umbilical cord blood as described herein and cultured in Iscove'smodified Dulbecco's medium supplemented with 200 ng/ml stem cell factorand 20% heat-inactivated fetal bovine serum. Plasmid DNA encoding theprotein or proteins of interest is incubated with branched or linearPEIs varying in size from 0.8 K to 750 K (Sigma Aldrich, Saint Louis,Mo., USA; Fermetas, Hanover, Md., USA). PEI is prepared as a stocksolution at 4.2 mg/ml distilled water and slightly acidified to pH 5.0using HCl. The DNA may be combined with the PEI for 30 min at roomtemperature at various nitrogen/phosphate ratios based on thecalculation that 1 μg of DNA contains 3 nmol phosphate and 1 μl of PEIstock solution contains 10 nmol amine nitrogen. The isolated CD34+ cellsare seeded with the DNA/cationic complex, centrifuged at 280×g for 5 minand incubated in culture medium for 4 or more h until gene expression isassessed.

A plasmid vector may be introduced into a hematopoietic stem cell usinga physical method such as particle-mediated transfection, “gene gun”,biolistics, or particle bombardment technology (Papapetrou, et al.,(2005) Gene Therapy 12:S118-S130). In this instance, DNA encoding theprotein and/or peptides of interest is absorbed onto gold particles andadministered to cells by a particle gun. This approach may be used, forexample, to transfect hematopoietic stem cells derived from umbilicalcord blood (See, e.g., Verma et al., Gene Therapy 5:692-699 (1998)). Assuch, umbilical cord blood is isolated and diluted three fold inphosphate buffered saline. CD34+ cells are purified using an anti-CD34monoclonal antibody in combination with magnetic microbeads coated witha secondary antibody and a magnetic isolation system (e.g., MiltenyiMiniMac System, Auburn, Calif., USA). The CD34+ enriched cells may becultured as described herein. Alternatively, the CD34+ enriched cellsmay be cultured on irradiated stromal cells in IMDM medium, for example,with 20% fetal bovine serum, 1% deionized bovine serum albumin,penicillin/streptomycin, L-glutamine, 2-mercaptoethanol andhydrocortisone supplemented with IL-3 (5 ng/ml), IL-6 (25 ng/ml), andstem cell factor (50 ng/ml). For transfection, plasmid DNA isprecipitated onto a particle, for example gold beads, by treatment withcalcium chloride and spermidine. Following washing of the DNA-coatedbeads with ethanol, the beads may be delivered into the cultured cellsusing, for example, a Biolistic PDS-1000/He System (Bio-Rad, Hercules,Calif., USA). A reporter gene such as, for example, beta-galactosidase,chloramphenicol acetyltransferase, luciferase, or green fluorescentprotein may be used to assess efficiency of transfection.

Alternatively, electroporation methods may be used to introduce aplasmid vector into hematopoietic stem cells (See, e.g., Wu et al., GeneTher. 8:384-390 (2001)). Electroporation creates transient pores in thecell membrane, allowing for the introduction of various molecules intothe cells including, for example, DNA and RNA as well as antibodies anddrugs. As such, CD34+ cells are isolated and cultured as describedherein. Immediately prior to electroporation, the cells are isolated bycentrifugation for 10 min at 250×g at room temperature and resuspendedat 0.2-10×10⁶ viable cells/ml in an electroporation buffer such as, forexample, X-VIVO 10 supplemented with 1.0% human serum albumin (HSA). Theplasmid DNA (1-50 μg) is added to an appropriate electroporation cuvettealong with 500 μl of cell suspension. Electroporation may be done using,for example, an ECM 600 electroporator (Genetronics, San Diego, Calif.,USA) with voltages ranging from 200 V to 280 V and pulse lengths rangingfrom 25 to 70 milliseconds. A number of alternative electroporationinstruments are commercially available and may be used for this purpose(e.g., Gene Pulser Xcell™, BioRad, Hercules, Calif.; Cellject Duo,Thermo Science, Milford, Mass.). Alternatively, efficientelectroporation of isolated CD34+ cells may be performed using thefollowing parameters: 4 mm cuvette, 1600 μF, 550 V/cm, and 10 μg of DNAper 500 μl of cells at 1×10⁵ cells/ml (Oldak et al., Acta BiochimicaPolonica 49:625-632 (2002)).

Nucleofection, a form of electroporation, may also be used to transfecthematopoietic stem cells. In this instance, transfection is performedusing electrical parameters in cell-type specific solutions that enableDNA (or other reagents) to be directly transported to the nucleus thusreducing the risk of possible degradation in the cytoplasm. For example,a Human CD34 Cell Nucleofector™ Kit (from amaxa inc.) may be used totransfect hematopoietic stem cells. In this instance, 1-5×10⁶ cells inHuman CD34 Cell Nucleofector™ Solution are mixed with 1-5 μg of DNA andtransfected in the Nucleofector™ instrument using preprogrammed settingsas determined by the manufacturer.

Hematopoietic stem cells may be non-virally transfected with aconventional expression vector which is unable to self-replicate inmammalian cells unless it is integrated in the genome. Alternatively,hematopoietic stem cells may be transfected with an episomal vectorwhich may persist in the host nucleus as autonomously replicatinggenetic units without integration into chromosomes (Papapetrou et al.,Gene Therapy 12:S118-S130 (2005)). These vectors exploit geneticelements derived from viruses that are normally extrachromosomallyreplicating in cells upon latent infection such as, for example, EBV,human polyomavirus BK, bovine papilloma virus-1 (BPV-1), herpes simplexvirus-1 (HSV) and Simian virus 40 (SV40). Mammalian artificialchromosomes may also be used for nonviral gene transfer (Vanderbyl etal., Exp. Hematol. 33:1470-1476 (2005)).

III. Loading of a Target-binding Agent or Molecular Agent into Red BloodCells

In an embodiment, red blood cells are loaded with one or moretarget-binding agents, such that the one or more target-binding agentsare internalized within the red blood cell. In an embodiment, red bloodcells are loaded with one or more molecular agents. A molecular agentmay include, but is not limited to, a compound that is configured toprovide an activity to the subject and/or to the red blood cellfollowing administration. In an embodiment, such agent may include, butis not limited to, one or more therapeutic agents or imaging agents.

A. Methods of Loading Red Blood Cells

A number of methods may be used to load modified red blood cells with anagent (e.g., target-binding agent or molecular agent) such as, forexample, hypotonic lysis, hypotonic dialysis, osmosis, osmotic pulsing,osmotic shock, ionophoresis, electroporation, sonication,microinjection, calcium precipitation, membrane intercalation, lipidmediated transfection, detergent treatment, viral infection, diffusion,receptor mediated endocytosis, use of protein transduction domains,particle firing, membrane fusion, freeze-thawing, mechanical disruption,and filtration (See, e.g., U.S. Pat. No. 6,495,351 B2; U.S. PatentApplication 2007/0243137 A1).

For hypotonic lysis, modified red blood cells are exposed to low ionicstrength buffer causing them to burst. The therapeutic agent such as anantibiotic or chemotherapeutic agent, for example, distributes withinthe cells. Red blood cells may be hypotonically lysed by adding 30-50fold volume excess of 5 mM phosphate buffer (pH 8), for example, to apellet of isolated red blood cells. The resulting lysed cell membranesare isolated by centrifugation. The pellet of lysed red blood cellmembranes is resuspended and incubated in the presence of thetherapeutic agent in a low ionic strength buffer for 30 min, forexample. Alternatively, the lysed red blood cell membranes may beincubated with the therapeutic agent for as little as one minute or aslong as several days, depending upon the best conditions determined toefficiently load the cells.

Alternatively, red blood cells may be loaded with a therapeutic agentusing controlled dialysis against a hypotonic solution to swell thecells and create pores in the cell membrane (See, e.g., U.S. Pat. Nos.4,327,710, 5,753,221, and 6,495,351 B2). For example, a pellet ofisolated red blood cells is resuspended in 10 mM HEPES, 140 mM NaCl, 5mM glucose pH 7.4 and dialyzed against a low ionic strength buffercontaining 10 mM NaH₂PO₄, 10 mM NaHCO₃, 20 mM glucose, and 4 mM MgCl₂,pH 7.4. After 30-60 min, the red blood cells are further dialyzedagainst 16 mM NaH₂PO₄, pH 7.4 solution containing the therapeutic agentfor an additional 30-60 min. All of these procedures may be optimallyperformed at a temperature of 4° C. In some instances, it may bebeneficial to load a large quantity of red blood cells with atherapeutic agent by a dialysis approach and as such a specificapparatus designed for this purpose may be used (See, e.g., U.S. Pat.Nos. 4,327,710, 6,139,836 and 6,495,351 B2).

The loaded red blood cells can be resealed by gentle heating in thepresence of a physiological solution such as, for example, 0.9% saline,phosphate buffered saline, Ringer's solution, cell culture medium, bloodplasma or lymphatic fluid. For example, well-sealed membranes may begenerated by treating the disrupted red blood cells for 1-2 min in 150mM salt solution of, for example, 100 mM phosphate (pH 8.0) and 150 mMsodium chloride at a temperature of 60° C. Alternatively, the cells maybe incubated at a temperature of 25-50° C. for 30 min to 4 h, forexample (See, e.g., U.S. Patent Application 2007/0243137 A1).Alternatively, the disrupted red blood cells may be resealed byincubation in 5 mM adenine, 100 mM inosine, 2 mM ATP, 100 mM glucose,100 mM Na-pyruvate, 4 mM MgCl₂, 194 mM NaCl, 1.6 M KCl, and 35 mMNaH₂PO₄, pH 7.4 at a temperature of 37° C. for 20-30 min (See, e.g.,U.S. Pat. No. 5,753,221).

For electroporation, for example, modified red blood cells are exposedto an electrical field which causes transient holes in the cellmembrane, allowing the therapeutic agent to diffuse into the cell (See,e.g., U.S. Pat. No. 4,935,223). Modified red blood cell are suspended ina physiological and electrically conductive media such as, for example,platelet-free plasma to which the therapeutic agent is added. Themixture in a volume ranging from 0.2 to 1.0 ml is placed in anelectroporation cuvette and cooled on ice for 10 min. The cuvette isplaced in an electroporation apparatus such as, for example, an ECM 830(from BTX Instrument Division, Harvard Apparatus, Holliston, Mass.). Thecells are electroporated with a single pulse of approximately 2.4milliseconds in length and a field strength of approximately 2.0 kV/cm.Alternatively, electroporation of red blood cells may be carried outusing double pulses of 2.2 kV delivered at 0.25 μF using a Bio-Rad GenePulsar apparatus (Bio-Rad, Hercules, Calif., USA) to achieve a loadingcapacity of over 60% (Flynn et al., Cancer Lett. 82:225-229 (1994)). Thecuvette is returned to the ice bath for 10-60 min and then placed in a37° C. water bath to induce resealing of the cell membrane.

For sonication, for example, modified red blood cells are exposed tohigh intensity sound waves, causing transient disruption of the cellmembrane allowing the therapeutic agent to diffuse into the cell.

For detergent treatment, for example, modified red blood cells aretreated with a mild detergent which transiently compromises the cellmembrane by creating holes, for example, through which the therapeuticagent may diffuse. After cells are loaded, the detergent is washed fromthe cells. For example, the detergent may be saponin.

For receptor mediated endocytosis, the modified red blood cell may havea surface receptor which upon binding of the therapeutic agents induceinternalization of the receptor and the associated therapeutic agent.

In an embodiment, the therapeutic agent may be loaded into a modifiedred blood cell by fusing or conjugating the agent to proteins and/orpeptides capable of crossing or translocating the plasma membrane (See,e.g., U.S. Patent Application 2002/0151004 A1). Examples of proteindomains and sequences that are capable of translocating a cell membraneinclude, for example, sequences from the HIV-1-transactivating protein(TAT), the Drosophila Antennapedia homeodomain protein, the herpessimplex-1 virus VP22 protein, and transportin, a fusion between theneuropeptide galanin and the wasp venom peptide mastoparan. As such, atherapeutic agent may be fused or conjugated to all or part of the TATpeptide, for example. A fusion protein containing all or part of the TATpeptide and the therapeutic agent such as an antibody, enzyme, orpeptide, for example, may be generated using standard recombinant DNAmethods. Alternatively, all or part of the TAT peptide may be chemicallycoupled to a functional group associated with the therapeutic agent suchas, for example, a hydroxyl, carboxyl or amino group. In some instances,the link between the TAT peptide and the therapeutic agent may be pHsensitive such that once the complex has entered the intracellularenvironment, the therapeutic agent is separated from the TAT peptide.

For mechanical firing, for example, the modified red blood cell may bebombarded with the therapeutic agent attached to a heavy or chargedparticle such as, for example, gold microcarriers and are mechanicallyor electrically accelerated such that they traverse the cell membrane.Microparticle bombardment of this sort may be achieved using, forexample, the Helios Gene Gun (from, e.g., Bio-Rad, Hercules, Calif.,USA).

Alternatively, the modified red blood cell may be loaded with atherapeutic agent by fusion with a synthetic vesicle such as, forexample, a liposome. In this instance, the vesicles themselves areloaded with the therapeutic agent using one or more of the methodsdescribed herein. Alternatively, the therapeutic agent may be loadedinto the vesicles during vesicle formation. The loaded vesicles are thenfused with the modified red blood cells under conditions that enhancecell fusion. Fusion of a liposome, for example, with a cell may befacilitated using various inducing agents such as, for example,proteins, peptides, polyethylene glycol (PEG), and viral envelopeproteins or by changes in medium conditions such as pH (See, e.g., U.S.Pat. No. 5,677,176).

For filtration, the modified red blood cell and the therapeutic agentmay be forced through a filter of pore size smaller than the red bloodcell causing transient disruption of the cell membrane and allowing thetherapeutic agent to enter the cell.

For freeze thawing, the modified red blood cells are sent throughseveral freeze thaw cycles, resulting in cell membrane disruption (See,e.g., U.S. Patent Application 2007/0243137 A1). In this instance, apellet of packed red blood cells (0.1-1.0 ml) is mixed with an equalvolume (0.1-1.0 ml) of an isotonic solution (e.g., phosphate bufferedsaline) containing the therapeutic agent. The red blood cells are frozenby immersing the tube containing the cells and therapeutic agent intoliquid nitrogen, for example. Alternatively, the cells may be frozen byplacing the tube in a freezer at −20° C. or −80° C., for example. Thecells are then thawed in a 23° C. water bath and the cycle repeated ifnecessary to increase loading.

The therapeutic agent may be selected from a variety of known smallmolecule pharmaceuticals. Alternatively, the therapeutic agent may be aninactivating peptide nuclei acid (PNA), an RNA or DNA oligonucleotideaptamer, an interfering RNA (iRNA), a peptide, or a protein.

The therapeutic agent may be loaded into the cell in a solubilized form.As such, the therapeutic agent is solubilized in an appropriate bufferprior to loading into red blood cells.

Alternatively, the therapeutic agent may be loaded into red blood cellsin a particulate form as a solid microparticulate (See, e.g., U.S.Patent Applications 2005/0276861 A1 and U.S. 2006/0270030 A1). In thisinstance, the therapeutic agent may be poorly water-soluble with asolubility of less than 1-10 mg/ml. As such, microparticles of less than10 μm may be generated using a variety of techniques such as, forexample, energy addition techniques such as milling (e.g., pearlmilling, ball milling, hammer milling, fluid energy milling, jetmilling), wet grinding, cavitation or shearing with a microfluidizer,and sonication; precipitation techniques such as, for example,microprecipitation, emulsion precipitation, solvent-antisolventprecipitation, phase inversion precipitation, pH shift precipitation,infusion precipitation, temperature shift precipitation, solventevaporation precipitation, reaction precipitation, compressed fluidprecipitation, protein microsphere precipitation; and other techniquessuch as spraying into cryogenic fluids (See, e.g., U.S. PatentApplication 2005/0276861 A1). Water soluble molecules may also be usedto form solid microparticles in the presence of various polymers suchas, for example, polylactate-polyglycolate copolymer (PLGA),polycyanoacrylate, albumin, and/or starch (See, e.g., U.S. PatentApplication 2005/0276861 A1). Alternatively, a water soluble moleculemay be encapsulated in a vesicle to form a microparticle. Themicroparticles composed of the therapeutic agent may be incorporatedinto a modified red blood cell using the methods described herein.

A modified red blood cell loaded with a therapeutic agent may beadministered intravenously, intramuscularly, subcutaneously,intradermally, intra-articularly, intrathecally, epidurally,intracerebrally, by buccal administration, rectally, topically,transdermally, orally, intranassaly, by pulmonary route,intraperitoneally, intra-opthalmically, or retro-orbitally. The cellsmay be administered by bolus injection, by intermittent infusion, or bycontinuous infusion, for example.

B. Molecular Agents

A variety of different agents may be loaded into red blood cells asdescribed above. It will be appreciated that it is not necessary for asingle agent to be used, and that it is possible to load two or moreagents into a cell. Accordingly, the term “agent” also includesmixtures, fusions, combinations and conjugates, of atoms, molecules,etc. as disclosed herein. For example, an agent may include, but is notlimited to, a nucleic acid combined with a polypeptide; two or morepolypeptides conjugated to each other; a protein conjugated to abiologically active molecule (which may be a small molecule such as aprodrug); or a combination of a biologically active molecule with animaging agent.

1. Therapeutic Agents

In an embodiment, the molecular agent is a therapeutic agent, such as asmall molecule drug or biological effector molecule. For example, thetherapeutic agent may be a biological effector molecule which hasactivity in a biological system. Biological effector molecules, include,but are not limited to, a protein, polypeptide, or peptide, including,but not limited to, a structural protein, an enzyme, a cytokine (such asan interferon and/or an interleukin), a polyclonal or monoclonalantibody, or an effective part thereof, such as an Fv fragment, whichantibody or part thereof, may be natural, synthetic or humanized, apeptide hormone, a receptor, or a signaling molecule. Included withinthe term “immunoglobulin” are intact immunoglobulins as well as antibodyfragments such as Fv, a single chain Fv (scFv), a Fab or a F(ab′)₂.

Therapeutic agents of interest include, without limitation,pharmacologically active drugs, genetically active molecules, etc.Therapeutic agents of interest include antineoplastic agents,anti-inflammatory agents, hormones or hormone antagonists, ion channelmodifiers, and neuroactive agents. Examples of of therapeutic agentsinclude those described in, “The Pharmacological Basis of Therapeutics,”Goodman and Gilman, McGraw-Hill, New York, N.Y., (1996), Ninth edition,under the sections: Drugs Acting at Synaptic and NeuroeffectorJunctional Sites; Drugs Acting on the Central Nervous System; Autacoids:Drug Therapy of Inflammation; Water, Salts and Ions; Drugs AffectingRenal Function and Electrolyte Metabolism; Cardiovascular Drugs; DrugsAffecting Gastrointestinal Function; Drugs Affecting Uterine Motility;Chemotherapy of Parasitic Infections; Chemotherapy of MicrobialDiseases; Chemotherapy of Neoplastic Diseases; Drugs Used forImmunosuppression; Drugs Acting on Blood-Forming organs; Hormones andHormone Antagonists; Vitamins, Dermatology; and Toxicology, allincorporated herein by reference. Also included are toxins, andbiological and chemical warfare agents, for example see Somani, S. M.(ed.), Chemical Warfare Agents, Academic Press, New York (1992)).

In an embodiment, the biological effector molecules are immunoglobulins,antibodies, Fv fragments, etc., that are capable of binding to antigensin an intracellular environment. These types of molecules are known as“intrabodies” or “intracellular antibodies.” An “intracellular antibody”or an “intrabody” includes an antibody that is capable of binding to itstarget or cognate antigen within the environment of a cell, or in anenvironment that mimics an environment within the cell. Selectionmethods for directly identifying such “intrabodies” include the use ofan in vivo two-hybrid system for selecting antibodies with the abilityto bind to antigens inside mammalian cells. Such methods are describedin PCT/GB00/00876, incorporated herein by reference. Techniques forproducing intracellular antibodies, such as anti-β-galactosidase scFvs,have also been described in Martineau et al., J Mol Biol 280:117-127(1998) and Visintin et al., Proc. Natl. Acad. Sci. USA 96:11723-1728(1999).

In an embodiment, the biological effector molecule includes, but is notlimited to, at least one of a protein, a polypeptide, a peptide, anucleic acid, a virus, a virus-like an amino acid, an amino acidanalogue, a modified amino acid, a modified amino acid analogue, asteroid, a proteoglycan, a lipid and a carbohydrate or a combinationthereof (e.g., chromosomal material comprising both protein and DNAcomponents or a pair or set of effectors, wherein one or more convertanother to active form, for example catalytically).

A biological effector molecule may include a nucleic acid, including,but not limited to, an oligonucleotide or modified oligonucleotide, anantisense oligonucleotide or modified antisense oligonucleotide, anaptamer, a cDNA, genomic DNA, an artificial or natural chromosome (e.g.,a yeast artificial chromosome) or a part thereof, RNA, including ansiRNA, a shRNA, mRNA, tRNA, rRNA or a ribozyme, or a peptide nucleicacid (PNA); a virus or virus-like particles; a nucleotide orribonucleotide or synthetic analogue thereof, which may be modified orunmodified.

The biological effector molecule can also be an amino acid or analoguethereof, which may be modified or unmodified or a non-peptide (e.g.,steroid) hormone; a proteoglycan; a lipid; or a carbohydrate. If thebiological effector molecule is a polypeptide, it can be loaded directlyinto a modified red blood cell, according to the methods describedherein. Alternatively, a nucleic acid molecule bearing a sequenceencoding a polypeptide, which sequence is operatively linked totranscriptional and translational regulatory elements active in a cellat a target site, may be loaded.

Small molecules, including inorganic and organic chemicals, may also beused. In an embodiment, the small molecule is a pharmaceutically activeagent. Useful classes of pharmaceutically active agents include, but arenot limited to, antibiotics, anti-inflammatory drugs, angiogenic orvasoactive agents, growth factors and chemotherapeutic (anti-neoplastic)agents (e.g., tumour suppressers).

If a prodrug is loaded in an inactive form, a second effector moleculemay be loaded into a modified red blood cell, or a red blood cell thatis to be modified according to the disclosure herein. Such a secondeffector molecule is usefully an activating polypeptide which convertsthe inactive prodrug to active drug form. In an embodiment, activatingpolypeptides include, but are not limited to, viral thymidine kinase(encoded by Genbank Accession No. J02224), carboxypeptidase A (encodedby Genbank Accession No. M27717), α-galactosidase (encoded by GenbankAccession No. M13571), β-gluucuronidase (encoded by Genbank AccessionNo. M15182), alkaline phosphatase (encoded by Genbank Accession No.J03252 J03512), or cytochrome P-450 (encoded by Genbank Accession No.D00003 N00003), plasmin, carboxypeptidase G2, cytosine deaminase,glucose oxidase, xanthine oxidase, β-glucosidase, azoreductase,t-gutamyl transferase, β-lactamase, or penicillin amidase.

Either the polypeptide or the gene encoding it may be loaded into themodified, or to-be-modified, red blood cells; if the latter, both theprodrug and the activating polypeptide may be encoded by genes on thesame recombinant nucleic acid construct. Furthermore, either the prodrugor the activator of the prodrug may be transgenically expressed inhematopoietic stem cells and already loaded into the red blood cell. Therelevant activator or prodrug (as the case may be) is then loaded as asecond agent according to the methods described herein.

2. Imaging Agents

The agent may be an imaging agent, by which term is meant an agent whichmay be detected, whether in vitro or in vivo in the context of a tissue,organ or organism in which the agent is located. Examples of agentsinclude those useful for imaging of tissues in vivo or ex vivo. Forexample, imaging agents, such as labeled antibodies which are specificfor defined molecules, tissues or cells in an organism, may be used toimage specific parts of the body by releasing from the loaded red bloodcells at a desired location using electromagnetic radiation. This allowsimaging agents which are not completely specific for the desired target,and which might otherwise lead to more general imaging throughout theorganism, to be used to image defined tissues or structures. Forexample, in an embodiment, an antibody which is capable of imagingendothelial tissue is used to image endothelial cells in lower bodyvasculature, such as in the lower limbs, by releasing the antibodyselectively in the lower body by applying ultrasound thereto. Theelectromagnetic energy will preferentially lyse the red blood cells inthe desired target site, thereby achieving selective therapeutic effectswith minimal damage to normal cells.

In an embodiment, the imaging agent emits a detectable signal, such asvisible light or other electromagnetic radiation. In another embodiment,the imaging agent is a radioisotope, for example ³²P or ³⁵S or ⁹⁹Tc, ora quantum dot, or a molecule such as a nucleic acid, polypeptide, orother molecule, conjugated with such a radioisotope. In an embodiment,the imaging agent is opaque to radiation, such as X-ray radiation. Inanother embodiment, the imaging agent comprises a targetingfunctionality by which it is directed to a particular cell, tissue,organ or other compartment within the body of an animal. For example,the agent may comprise a radiolabelled antibody which specifically bindsto defined molecule(s), tissue(s) or cell(s) in an organism.

In an embodiment, the imaging agent is a contrast dye. For example, anMRI contrast agent can comprise a paramagnetic contrast agent (such as agadolinium compound), a superparamagnetic contrast agent (such as ironoxide nanoparticles), a diamagnetic agent (such as barium sulfate), andcombinations thereof. Metal ions preferred for MRI include those withatomic numbers 21-29, 39-47, or 57-83, and, more typically, aparamagnetic form of a metal ion with atomic numbers 21-29, 42, 44, or57-83. Particularly preferred paramagnetic metal ions are selected fromthe group consisting of Gd(III), Fe(III), Mn(II and III), Cr(III),Cu(II), Dy(III), Tb(III and IV), Ho(III), Er(III), Pr(III) and Eu(II andIII). Gd(III) is particularly useful. Note that as used herein, the term“Gd” is meant to convey the ionic form of the metal gadolinium; such anionic form can be written as GD(III), GD3+, etc. with no difference inionic form contemplated. A CT contrast agent can comprise iodine (ionicor non-ionic formulations), barium, barium sulfate, Gastrografin (adiatrizoate meglumine and diatrizoate sodium solution), and combinationsthereof. In another embodiment, a PET or SPECT contrast agent cancomprise a metal chelate.

IV. Incorporating Positive Marker(s) into Modified Red Blood Cells

The modified red blood cells may also be labeled with one or morepositive markers that can be used to monitor over time the number orconcentration of modified red blood cells in the blood circulation of anindividual. It is anticipated that the overall number of modified redblood cells will decay over time following initial transfusion. As such,it may be appropriate to correlate the signal from one or more positivemarkers with that of the activated molecular marker, generating aproportionality of signal that will be independent of the number ofmodified red blood cells remaining in the circulation. There arepresently several fluorescent compounds, for example, that are approvedby the Food & Drug Administration for human use including but notlimited to fluorescein, indocyanin green, and rhodamine B. For example,red blood cells may be non-specifically labeled with fluoresceinisothiocyanate (FITC; Bratosin et al., Cytometry 46:351-356 (2001)). Asolution of FITC-labeled lectins in phosphate buffered saline (PBS) with0.2 mM phenylmethysulfonyl fluoride (PMSF) is added to an equal volumeof isolated red blood cells in the same buffer. The cells are incubatedwith the FITC-labeled lectins for 1 h at 4° C. in the dark. The lectinsbind to sialic acids and beta-galactosyl residues on the surface of thered blood cells.

It is anticipated that other dyes may be useful for tracking modifiedred blood cells in human and non-human circulation. A number of reagentsmay be used to non-specifically label a red blood cell. For example redblood cells may be labeled with PKH26 Red (See, e.g., Bratosin, et al.,(1997) Cytometry 30:269-274). Red blood cells (1-3×10⁷ cells) aresuspended in 1 ml of “diluent C” and rapidly added to 1 ml or 2 μM PKH26dissolved in “diluent C”. The mixture is mixed by gentle pipetting andincubated at 25° C. for 2-5 min with constant stirring. The labeling maybe stopped by adding an equal volume of human serum or compatibleprotein solution (e.g, 1% bovine serum albumin). After an additionalminute, an equal volume of cell culture medium is added and the cellsare isolated by centrifugation at 2000×g for 5 min, for example. Cellsare washed three times by repeated suspension in cell culture medium andcentrifugation. PHK26-labeled cells may be monitored with a maximumexcitation wavelength of 551 nm and a maximum emission wavelength of 567nm.

VivoTag 680 (VT680; VisEn Medical, Woburn, Mass., USA) may be used totrack cells in vivo. VT680 is a near-infrared fluorochrome with a peakexcitation wavelength of 670±5 nm and a peak emission wavelength of688±5 nm. VT680 also contains an amine reactive NHS ester which enablesit to cross-link with proteins and peptides. As such, the surface ofcells may be labeled with VT680 (See, e.g., Swirski, et al., (2007) PloSONE 10:e1075). For example, 4×10⁶ cells/ml are incubated with VT680diluted in complete culture medium at a final concentration of 0.3 to300 μg/ml for 30 min at 37° C. The cells are washed twice with completeculture medium after labeling. Cells may be non-specifically labeledbased on proteins expressed on the surface of the modified red bloodcell. Alternatively, a specific protein may be labeled with VT680. Insome instances, an antibody directed against a specific proteinassociated with the modified red blood cell may be used to selectivelylabel cells. In other instances, a protein or peptide may be directlylabeled with VT680 ex vivo and subsequently either attached to thesurface of the cell or incorporated into the interior of the cell usingmethods described here in for the uptake of nucleic acids.

In vivo monitoring, for example, may be performed using, for example,the dorsal skin fold. As such, laser scanning microscopy may beperformed using, for example, an Olympus IV 100 in which VT680 isexcited with a red laser diode of 637 nm and detected with a 660/LPfilter. Alternatively, multiphoton microscopy may be performed using,for example, a BioRad Radiance 2100 MP centered around an Olympus BX51equipped with a 20×/0.95 NA objective lens and a pulsed Ti:Sapphirelaser tuned to 820 nm. The latter wavelength is chosen because VT680 hasa peak in its two-photon cross-section at 820 nm.

Alternatively, a modified red blood cell may be labeled with other redand/or near-infrared dyes including, for example, cyanine dyes such asCy5, Cy5.5, and Cy7 (Amersham Biosciences, Piscataway, N.J., USA) and/ora variety of Alexa Fluor dyes including Alexa Fluor 633, Alexa Fluor635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700and Alexa Fluor 750 (Molecular Probes-Invitrogen, Carlsbad, Calif.,USA). Additional fluorophores include IRD41 and IRD700 (LI-COR, Lincoln,Nebr., USA), NIR-1 and 1C5-OSu (Dejindo, Kumamotot, Japan), LaJolla Blue(Diatron, Miami, Fla., USA), FAR-Blue, FAR-Green One, and FAR-Green Two(Innosense, Giacosa, Italy), ADS 790-NS and ADS 821-NS (American DyeSource, Montreal, Calif.). Quantum dots (Qdots) of variousemission/excitation properties may also be used for labeling cells (See,e.g., Jaiswal et al., Nature Biotech. 21:47-51 (2003)). Many of thesefluorophores are available from commercial sources either attached toprimary or secondary antibodies or as amine-reactive succinimidyl ormonosuccinimidyl esters, for example, ready for conjugation to a proteinor proteins either on the surface or inside the red blood cells.

Magnetic nanoparticles may be used to track cells in vivo using highresolution MRI (Montet-Abou et al., Molecular Imaging 4:165-171 (2005)).Magnetic particles may be internalized by several mechanisms. Magneticparticles may be taken up by a cell through fluid-phase pinocytosis orphagocytosis. Alternatively, the magnetic particles may be modified tocontain a surface agent such as, for example, the membrane translocatingHIV that peptide which promotes internalization. In some instances, amagnetic nanoparticle such as, for example, Feridex IV®, an FDA approvedmagnetic resonance contrast reagent, may be internalized intohematopoietic cells in conjunction with a transfection agent such as,for example, protamine sulfate (PRO), polylysine (PLL), andlipofectamine (LFA).

V. Formulations of Pharmaceutical Compositions

The modified red blood cells can be incorporated into pharmaceuticalcompositions suitable for administration. The pharmaceuticalcompositions generally comprise substantially purified modified redblood cells and a pharmaceutically-acceptable carrier in a form suitablefor administration to a subject. Pharmaceutically-acceptable carriersare determined in part by the particular composition being administered,as well as by the particular method used to administer the composition.Accordingly, there is a wide variety of suitable formulations ofpharmaceutical compositions for administering the antibody compositions(See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co.,Easton, Pa. 18^(th) ed. (1990)). The pharmaceutical compositions aregenerally formulated as sterile, substantially isotonic and in fullcompliance with all Good Manufacturing Practice (GMP) regulations of theU.S. Food and Drug Administration.

The terms “pharmaceutically-acceptable,” “physiologically-tolerable,”and grammatical variations thereof, as they refer to compositions,carriers, diluents and reagents, are used interchangeably and includematerials are capable of administration to or upon a subject without theproduction of undesirable physiological effects to a degree that wouldprohibit administration of the composition. For example,“pharmaceutically-acceptable excipient” includes an excipient that isuseful in preparing a pharmaceutical composition that is generally safe,non-toxic, and desirable, and includes excipients that are acceptablefor veterinary use as well as for human pharmaceutical use. Suchexcipients can be solid, liquid, semisolid, or, in the case of anaerosol composition, gaseous.

Examples of such carriers or diluents include, but are not limited to,water, saline, Ringer's solutions, dextrose solution, and 5% human serumalbumin. The use of such media and compounds for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or compound is incompatible with the modified red blood cells, usethereof in the compositions is contemplated. Supplementary activecompounds can also be incorporated into the compositions.

A pharmaceutical composition is formulated to be compatible with itsintended route of administration. The modified red blood cells can beadministered by parenteral, topical, intravenous, oral, subcutaneous,intraarterial, intradermal, transdermal, rectal, intracranial,intraperitoneal, intranasal; intramuscular route or as inhalants. Themodified red blood cells can optionally be administered in combinationwith other agents that are at least partly effective in treating variousdiseases including various actin- or microfilament-related diseases.

Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial compounds such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating compounds such as ethylenediaminetetraacetic acid (EDTA);buffers such as acetates, citrates or phosphates, and compounds for theadjustment of tonicity such as sodium chloride or dextrose. The pH canbe adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, e.g., water,ethanol, polyol (e.g., glycerol, propylene glycol, and liquidpolyethylene glycol, and the like), and suitable mixtures thereof. Theproper fluidity can be maintained, e.g., by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalcompounds, e.g., parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like. In many cases, it will be preferable toinclude isotonic compounds, e.g., sugars, polyalcohols such as manitol,sorbitol, sodium chloride in the composition. Prolonged absorption ofthe injectable compositions can be brought about by including in thecomposition a compound which delays absorption, e.g., aluminummonostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating themodified red blood cells in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired. Generally, dispersions are prepared by incorporating themodified red blood cells into a sterile vehicle that contains a basicdispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. The modified red blood cells can be administered inthe form of a depot injection or implant preparation which can beformulated in such a manner as to permit a sustained or pulsatilerelease of the active ingredient.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, themodified red blood cells can be incorporated with excipients and used inthe form of tablets, troches, or capsules. Oral compositions can also beprepared using a fluid carrier for use as a mouthwash, wherein thecompound in the fluid carrier is applied orally and swished andexpectorated or swallowed. Pharmaceutically compatible bindingcompounds, and/or adjuvant materials can be included as part of thecomposition. The tablets, pills, capsules, troches and the like cancontain any of the following ingredients, or compounds of a similarnature: a binder such as microcrystalline cellulose, gum tragacanth orgelatin; an excipient such as starch or lactose, a disintegratingcompound such as alginic acid, Primogel, or corn starch; a lubricantsuch as magnesium stearate or Sterotes; a glidant such as colloidalsilicon dioxide; a sweetening compound such as sucrose or saccharin; ora flavoring compound such as peppermint, methyl salicylate, or orangeflavoring.

For administration by inhalation, the modified red blood cells aredelivered in the form of an aerosol spray from pressured container ordispenser which contains a suitable propellant, e.g., a gas such ascarbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, e.g., fortransmucosal administration, detergents, bile salts, and fusidic acidderivatives. Transmucosal administration can be accomplished through theuse of nasal sprays or suppositories. For transdermal administration,the modified red blood cells are formulated into ointments, salves,gels, or creams as generally known in the art.

The modified red blood cell an also be prepared as pharmaceuticalcompositions in the form of suppositories (e.g., with conventionalsuppository bases such as cocoa butter and other glycerides) orretention enemas for rectal delivery.

In an embodiment, the modified red blood cells are prepared withcarriers that will protect the modified red blood cells against rapidelimination from the body, such as a controlled release formulation,including implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Methods for preparation of suchformulations will be apparent to those skilled in the art. The materialscan also be obtained commercially from Alza Corporation and NovaPharmaceuticals, Inc. Liposomal suspensions (including liposomestargeted to infected cells with monoclonal antibodies to viral antigens)can also be used as pharmaceutically-acceptable carriers. These can beprepared according to methods known to those skilled in the art, e.g.,as described in U.S. Pat. No. 4,522,811.

Methods of Use I. General Methods of Targeting Cells or Tissues

This section will generally describe an embodiment of the methods ofusing the modified red blood cell compositions. Further details ofparticular applications are described in the sections that follow.

In one aspect, the disclosure provides methods of using a modified redblood cell to bind a target molecule, thereby localizing the modifiedred blood cell to a particular location (e.g., tissue or cell) within asubject. In an embodiment, the fusion protein is configured tofacilitiate fusion of the red blood cell with the target cell. In otherembodiments, upon binding of the target recognition moiety to the targetmolecule, the complex becomes activated. As such, a singlet oxygenmolecule can be delivered to the particular location (e.g., tissue orcell) by exposing the area to light of a suitable wavelength. Thedisclosure also provides for methods of using a modified red blood cellto bring a target cell or tissue in contact with a molecular agent,which is carried by the modified red blood cell. In an embodiment, themodified red blood cells may be useful for the treatment of infection(e.g., bacterial, fungal, viral or parasitic) or for the treatment ofcancer or other hyperproliferative disorders (e.g., restenosis or benignprostatic hyperplasia), by damaging or destroying the target cells.

As shown in FIG. 1, a target-binding agent 100 is composed of a targetrecognition moiety 105, a photoactivatable molecule 110, and a quenchermolecule 115. In the absence of a target, the photoactivatable molecule110 is in close proximity to the quencher molecule 115 and is notresponsive by light. In the presence of a target 120, an activated unit125 is generated. As such, the target recognition moiety 105 undergoes aconformational change 130. The quencher molecule 115 moves away from thephotoactivatable molecule 110, resulting in an activatedphotoactivatable molecule 135. In response to light energy 140, theactivated photoactivatable molecule 135 emits a reactive singlet oxygen145.

As shown in FIG. 2, one or more red blood cells 150 may be modified witha target-binding agent 100 to form a modified red blood cell. Themodified red blood cells can be released into the blood circulation ofthe subject. In circulation, the one or more modified red blood cells150 may come into contact with a target cell 160 which expresses on itssurface, for example, a target 120 recognized by the target recognitionmoiety of the target-binding agent. The target cell 160 may be apathogen such as for example a bacterium, a fungus, or a parasite.Alternatively, the target cell 160 may be a cancerous cell such as forexample a leukemia cell, a circulating tumor cell (CTC), or othercancerous cell. Upon binding, the target-binding agent is converted intoan activated unit. In response to light energy 140, the activated unit125 releases a reactive singlet oxygen 145.

As shown in FIG. 3, one or more red blood cells 150 may be modified withone or more target-binding agents 100 to form a modified red blood cell.Similarly, one or more target cells 160 may have one or more targets 120recognized by the target-binding agent 100. As such, one or more targets120 may bind to one or more target-binding agents 100 to generate one ormore activated units 125.

As shown in FIG. 4, one or more modified red blood cells 150 modifiedwith one or more target-binding agents 100 may interact with one or moretarget cells 160 with one or more targets 120 recognized by thetarget-binding agent 100. As such, one or more modified red blood cells150 may interact with one or more target cells. Singlet oxygen mayproduce cellular damage. Since the singlet oxygen has a very shortlifetime (microseconds), the photodamage can be expected to be within ashort radius of the target molecule.

As shown in FIG. 5, upon binding of a modified red blood cell 150 to atarget cell 160, the target-binding agent is converted into theactivated unit 125. The activated unit 125 may now be excited by lightenergy 135 resulting in release of a reactive singlet oxygen 145. Thereactive singlet oxygen 145 may, in turn, interact with the target cell160. The reactive singlet oxygen 145 may cause apoptosis and/or necrosisof the target cell 160 leading to a dead or inactivated target cell 170.

As shown in FIG. 6, in some instances, the modified red blood cells 150may be loaded with a therapeutic agent 180. A therapeutic agent 180 maybe a small molecule drug, a biological drug such as, for example, anantibody, a ligand, a receptor and/or an enzyme, or an oligonucleotidesuch as, for example, an RNA or DNA aptamer, an interfering RNA, and/oran antisense RNA. Upon binding of a modified red blood cell 150 to atarget cell 160, the target-binding agent is converted into theactivated unit 125. The activated unit 125 may now be excited by lightenergy 135 resulting in release of a reactive singlet oxygen 145. Thereactive singlet oxygen 145 may, in turn, interact with the modified redblood cell 150. The reactive singlet oxygen 145 may cause apoptosisand/or necrosis of the modified red blood cell 150 resulting in a deador inactivated red blood cell 190. The inactivated modified red bloodcell 190 releases the therapeutic agent through the compromised cellmembrane 200 in proximity of the target cell 160.

As shown in FIG. 7, in some instances, the modified red blood cells 150comprising one or more target-binding agents 100 may also be modifiedwith another target recognition moiety 210. The target recognitionmoiety 210 may recognize a receptor 220 on the target cell 160. Thetarget recognition moiety 210 and the receptor 220 may be an antibodyand an antigen, respectively. Alternatively, the target recognitionmoiety 210 and the receptor 220 may be a ligand/receptor pair. As such,modified red blood cells 150 and one or more target cells 160 mayinteract through the target-binding agent 100 and the target 120 to formthe activated unit 125, through the target recognition moiety 210 andthe receptor 220, or through a combination of both to facilitate a moreselective interaction, for example.

As shown in FIG. 8, in some instances, one or more target-binding agents100 may be loaded into the cytoplasm, for example, of one or more redblood cells 150 to form a modified red blood cell. The modified redblood cell may be further modified with another target recognitionmoiety 210. The target recognition moiety 210 may recognize a receptor220 on the target cell 160. In some instances, the interaction of thetarget recognition moiety 210 and the receptor 220 may lead to fusionand/or invasion of the red blood cell 150 by the target cell 160. Assuch, the modified red blood cell 150 may take up the target cell 160.Inside the modified red blood cell 150, the target cell 120 may interactwith the target-binding agent 100 to generate the activated unit 125.The internalized target cell 160 may then be damaged or destroyed whenthe activated unit is exposed to light of an appropriate wavelength andpower and a singlet oxygen radical molecule is produced.

As shown in FIG. 9, in some instances, the modified red blood cells 150modified with one or more target-binding agent 100 may also be modifiedwith another target recognition moiety 210. The target recognitionmoiety 210 may recognize a receptor 220 on the target cell 160. In someinstances, the interaction of the target recognition moiety 210 and thereceptor 220 may lead to fusion of the red blood cell 150 and the targetcell 160. Inside the target cell 160, the target-binding agent 100 mayinteract with the target 120 to generate the activated unit 125. Uponradiation with light energy 135, the activated unit 125 emits reactivesinglet oxygen leading to a damaged or destroyed target cell 170 and/ordamaged or destroyed red blood cell 190.

As shown in FIG. 10, in some instances, the modified red blood cell 150modified with one or more target-binding agent 100 and one or moretarget recognition moiety 210 may be loaded with a therapeutic agent180. The target recognition moiety 210 may recognize a receptor 220 onthe target cell 160. In some instances, the interaction of the targetrecognition moiety 210 and the receptor 220 may lead to fusion of thered blood cell 150 and the target cell 160. Inside the target cell 160,the target-binding agent 100 may interact with the target 120 togenerate the activated unit 125. Upon radiation with light energy 135,the activated unit 125 emits reactive singlet oxygen leading to damagedor destroyed red blood cell 190 and release of the therapeutic agent 180into the cytoplasm of the target cell 160.

A. Excitation of the Photoactivatable Molecule

In an embodiment, the one or more methods optionally include providingelectromagnetic energy to the subject, where the electromagnetic energyis configured to induce a response from the photoactivatable moleculesassociated with the modified red blood cells. In illustrativeembodiments, excitation of the one or more photoactivatable moleculesdirectly and/or indirectly damages the target cell and/or the red bloodcell.

In illustrative embodiments, the electromagnetic energy includes, but isnot limited to, one or more frequencies having one or morecharacteristics that taken as a whole are not considered unduly harmfulto the subject. In illustrative examples, such electromagnetic energymay include frequencies optionally including visible light (detected bythe human eye between approximately 400 nm and 700 nm) as well asinfrared (longer than 700 nm) and limited spectral regions ofultraviolet light, such as UVA light (between approximately 320 nm and400 nm). Electromagnetic energy includes, but is not limited to, singlephoton electromagnetic energy, two photon electromagnetic energy,multiple wavelength electromagnetic energy, and extended-spectrumelectromagnetic energy.

Electromagnetic energy may be configured as a continuous beam or as atrain of short pulses. In the continuous wave mode of operation, theoutput is relatively consistent with respect to time. In the pulsed modeof operation, the output varies with respect to time, optionally havingalternating “on” and “off” periods. Electromagnetic energy may beprovided by one or more lasers, for example, having one or more of acontinuous or pulsed mode of action. One or more pulsed lasers mayinclude, but are not limited to, Q-switched lasers, mode locking lasers,and pulsed-pumping lasers. Mode locked lasers emit extremely shortpulses on the order of tens of picoseconds down to less than 10femtoseconds, the pulses optionally separated by the time that a pulsetakes to complete one round trip in the resonator cavity. Due to theFourier limit, a pulse of such short temporal length may have a spectrumwhich contains a wide range of wavelengths.

In an embodiment, the electromagnetic energy is focused at a depth ofapproximately 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm,0.8 mm, 0.9 mm, 1.0 mm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6 mm,1.7 mm, 1.8 mm, 1.9 mm, 2.0 mm, 2.1 mm, 2.2 mm, 2.3 mm, 2.4 mm, 2.5 mm,2.6 mm, 2.7 mm, 2.8 mm, 2.9 mm, or 3.0 mm below the surface of the skin,beyond the surface of a wall of a blood vessel (e.g., in the bloodvessel lumen), and/or beyond a surface of an internal location. In anembodiment, the electromagnetic energy is focused at a depth ofapproximately 0.1 to 3 mm, 0.1 to 2.5 mm, 0.1 to 2.0 mm, 0.1 to 1.5 mm,0.1 to 1.0 mm, 0.1 to 0.5 mm, 0.5 to 3.0 mm, 0.5 to 2.5 mm, 0.5 to 2.0mm, 0.5 to 1.5 mm, 0.5 to 1.0 mm, 1.0 to 3.0 mm, 1.0 to 2.5 mm, 1.0 to2.0 mm, 1.0 to 1.5 mm, 1.5 to 3.0 mm, 1.5 to 2.5 mm, 1.5 to 2.0 mm, 2.0to 3.0 mm, 2.0 to 2.5 mm, or 2.5 to 3.0 mm below the surface of theskin, beyond the surface of a wall of a blood vessel (e.g., in the bloodvessel lumen), and/or beyond a surface of an internal location.

In an embodiment, the electromagnetic energy is generated by two photonshaving the same wavelength or substantially the same wavelength. In anembodiment, the electromagnetic energy is generated by sets of twophotons having different wavelengths. Electromagnetic energy at theenergy levels of the two photons is optionally focused at a depth belowthe surface of the skin, beyond the surface of a wall of a blood vessel(e.g., in the blood vessel lumen), and/or beyond a surface of aninternal location, and/or optionally at one or more depths. As usedherein, the term “two-photon” may include excitation optionally usingone or more femtosecond lasers. In an embodiment, two photonelectromagnetic energy is coupled through a virtual energy level and/orcoupled through an intermediate energy level.

As used herein, the term “extended-spectrum” may include a range ofpossible electromagnetic radiation wavelengths within the full spectrumof possible wavelengths, optionally from extremely long to extremelyshort and optionally including wide spectrum and narrow spectrumwavelengths.

In an embodiment, the electromagnetic energy may be defined spatiallyand/or directionally. In an embodiment, the electromagnetic energy maybe spatially limited, optionally spatially focused and/or spatiallycollimated. In an embodiment, the electromagnetic energy may bedirectionally limited, directionally varied, and/or directionallyvariable. In illustrative embodiments, the electromagnetic energyoptionally contacts less than an entire possible area, or less than anentire possible target, and/or is limited to a certain depth within atissue. In illustrative embodiments, the electromagnetic energy isspatially and/or directionally limited so that only areas approximatelybounded by the walls of one or more blood vessels are provided withelectromagnetic energy. In illustrative embodiments, the electromagneticenergy may be provided over an entire field (e.g., scanning acrossand/or the length of a blood vessel lumen), through movement of theelectromagnetic source, and/or through illumination from more than one,two, three, four, and/or multiple sources in the device. Alternatively,in some approaches illumination may be provided over less than an entirefield, for example, by illuminating according to a vector scanningapproach. In such approaches, illumination energy may be directed toless than all of the area, e.g., primarily in and/or around vascularregions or in areas of interest, such as areas where blood components ofinterest may be suspected to be or predicted to be. Alternatively, suchillumination of less than the entire region may be implemented by ascanning pattern encompassing the entire region combined with activatingthe source of electromagnetic energy only in selected locations.

B. Methods for Disrupting Modified Red Blood Cells Loaded with MolecularAgents

A modified red blood cell loaded with a molecular agent (e.g., atherapeutic agent) may be targeted to a specific pathogen or cell usingthe methods described herein. Upon interacting with the target cell, themodified red blood cell may be induced to release the therapeutic agent.There are a number of methods described for controlled release of atherapeutic agent from a red blood cell such as, for example, normal redblood cell break down, accelerated red blood cell breakdown due, forexample, to incompatible cells from different individuals or species,inappropriate blood type, and/or introduction of immunogenic protein onthe surface of the red blood cell, administering energy to selectivelydisrupt red blood cells such as, for example, ultrasound,radiofrequency, microwave, and/or infrared, incorporation of an enzymethat digests the cell membrane from the inside out, addition of a secondagent added at a specific time the initiates cell breakdown, and use ofthe complement system (See, e.g., U.S. Patent Application 2007/0243137A1).

In some instances, it may be of benefit to target macrophages with themodified red blood cells. Under normal circumstances, aged and/ordamaged red blood cells are cleared from circulation by macrophagephagocytosis. This process may be enhanced by artificially clusteringthe modified red blood cell transmembrane proteins using, for example,ZnCl₂ and bissulfosuccinimideilsuberate (BS³; See, e.g., U.S. Pat. No.6,139,836). In this instance, the modified red blood cells are treatedwith 1 mM ZnCl₂ in saline solution to cluster the proteins andsubsequently treated with BS³ for 15 min to irreversibly cross-link theclustered proteins.

A modified red blood cell may be loaded with metal particles which uponinteraction with an external energy source preferentially causes themodified red blood cells to be disrupted (See, e.g., U.S. Pat. No.6,645,464). As such, modified red blood cells may be loaded withcolloidal gold or gold clusters (1-20 nm in size) using hypotonic lysisin 5 mM phosphate buffer (pH 8) with 10 μM magnesium sulfate at atemperature of 4° C. In some instances, the modified red blood cells maybe simultaneously loaded with metal particles and a therapeutic agent,for example. The cells are resealed by warming to 37° C. for 5-15 min inthe presence of 0.2 M NaCl, for example. In some instances, it may bebeneficial to add small nucleating metal particles to a modified redblood cell and subsequently enlarging the particles in situ (See, e.g.,U.S. Pat. No. 6,645,464). As such, modified red blood cells that havebeen loaded with small nucleating metal particles and resealed may befurther treated with an autometallographic developer solution containinggold ions, for example, to generate large internal metal particles. Themodified red blood cells may be targeted to a specific tissue bed suchas, for example, a tumor, and subsequently irradiated to disrupt themodified red blood cell and release the loaded therapeutic agent.

C. Monitoring Interaction of Modified Red Blood Cell with TargetMolecule

In an embodiment, it may be useful to monitor the interaction of themodified red blood cells with a target molecule or target cell prior tothe exposure of a subject to light of a suitable wavelength. Theinteraction may be monitored by providing a modified red blood cellwhich includes a signaling molecule that is detectable upon binding ofthe modified red blood cell to the target cell or molecule.

In an embodiment, red blood cells may be modified with an aptamer-basedmolecular beacon to detect interaction of the modified red blood cellwith a target cell. RNA or DNA oligonucleotide-based aptamers incombination with fluorescent tags, for example, may be used as molecularbeacons to detect interactions between a modified red blood cell andmolecules on the surface of a pathogen and/or cancerous cell. Aptamersspecific for virtually any class of molecules may be isolated from alarge library of 10¹⁴ to 10¹⁵ random oligonucleotide sequences using aniterative in vitro selection procedure often termed “systematicevolution of ligands by exponential enrichment” (SELEX; Cao et al.,Current Proteomics 2:31-40 (2005); Proske et al., Appl. Microbiol.Biotechnol. 69:367-374 (2005)).

Molecular beacons may be dual labeled aptamer probes with a donorfluorophore at one end and an acceptor fluorophore or quencher at theother end. Upon binding of a specific target, the aptamer is configuredto undergo a conformational shift such that the distance between thedonor fluorophore and the acceptor fluorophore or quencher is altered,leading to a change in detectable fluorescence. This phenomenon isreferred to as fluorescence resonance energy transfer (FRET). FRET is adistance-dependent interaction between the electronic excited states oftwo dye molecules in which excitation is transferred from a donormolecule to an acceptor molecule without emission of a photon. In someinstances, interaction of a donor molecule with an acceptor molecule maylead to a shift in the emission wavelength associated with excitation ofthe acceptor molecule. In other instances, interaction of a donormolecule with an acceptor molecule may lead to quenching of the donoremission. As such, an aptamer-based molecular beacon may be used tomonitor changes in the fluorescent properties of the aptamer-basedmolecular beacon in response to binding a chemical entity such as, forexample, a molecule on the surface of a target cell.

A variety of donor and acceptor fluorophore pairs may be considered forFRET associated with an aptamer-based molecular beacon including, butnot limited to, fluorescein and tetramethylrhodamine; IAEDANS andfluorescein; fluorescein and fluorescein; and BODIPY FL and BODIPY FL. Anumber of Alexa Fluor (AF) fluorophores (from MolecularProbes-Invitrogen, Carlsbad, Calif., USA) may be paired with other AFfluorophores for use in FRET. Some examples include AF 350 with AF 488;AF 488 with AF 546, AF 555, AF 568, or AF 647; AF 546 with AF 568, AF594, or AF 647; AF 555 with AF594 or AF647; AF 568 with AF6456; andAF594 with AF 647.

Red blood cells may be modified with a cell-surface receptor thatsignals either directly or indirectly in response to ligand binding. Asan example, a G-protein-coupled receptor (GPCR) associated with amodified red blood cell may be used as an activatable molecular markerto monitor binding of a modified red blood cell to a target. The vastmajority of GPCRs internalize from the cell surface into acidicendosomes in response to agonist challenge (Milligan, DDT 8:579-585(2003)). As such, a GPCR may be labeled with a pH sensitive dye whichupon entering the acidic environment of the endosome changes itsemission properties. The GPCR may be labeled with CypHer5™, for example,which is a red-excited, pH-sensitive cyanine dye that is non-fluorescentat pH 7.4 and maximally fluorescent at pH 5.5 and is ideally suited formonitoring internalization of GPCRs (available from AmershamBiosciences, Piscataway, N.J., USA). CypHer5™ may be attached to aprotein by conjugation of CypHer5™ mono NHS ester to amine groups on thesurface of the protein (See, e.g., Adie et al., Biotechniques33:1152-1157 (2002)). In the case of labeling a GPRC or other cellsurface receptor, the receptor may be directly labeled with CypHer5™.Alternatively, a GPCR may be indirectly labeled by interaction with aCypHer5™ labeled antibody specific for that receptor (See, e.g., Adie etal., Biotechniques 33:1152-1157 (2002)). CypHer5™ signaling is monitoredat an emission wavelength of 695 nm using an excitation wavelength of633 nm. Other pH sensitive dyes that might be used for labeling a GPCRor other cell surface receptor include but are not limited tofluoroscein isothiocyanate (FITC), 1,4(and 5)-benzenedicarboxylic acid,2-[10-(dimethylamino)-4-fluoro-3-oxo-3H-benzo[c]xanthen-7-yl] (carboxySNARF-4F), 2′,7′-Bis(2-carboxylethyl)-5(6)-carboxyfluorescein (BCECF).

II. Methods of Treatment

In an aspect, one or more methods of treatment include providing one ormore modified red blood cells to a subject; wherein the one or moremodified red blood cells are associated with one or more targetrecognition moieties. In an aspect, one or more methods of treatmentinclude providing one or more modified red blood cells to a subject;wherein the one or more modified red blood cells include one or moretarget recognition moieties. In an embodiment, the target recognitionmoieties are designed to recognize one or more neoplastic cells orpathogens. In an embodiment, activation of the target-binding agent andsubsequent excitation of the photoactivatable molecule may cause releaseof a therapeutic agent (e.g., an antibiotic or chemotherapeutic agent)from the modified red blood cell. In other embodiments, the modified redblood cells include one or more fusion molecules that are designed toparticipate in a fusion of the modified red blood cells with the targetcells.

Briefly, the modified red blood cells are administered to the subjectbefore the target tissue, target composition or subject is subjected toelectromagnetic radiation. The composition may be administered in apharmaceutical formulation as described above. The dose of the modifiedred blood cells for an optimal therapeutic benefit can be determinedclinically. A certain length of time is allowed to pass for thecirculating or locally delivered modified red blood cells to be taken upby the target tissue. The unbound modified red blood cells are clearedfrom the circulation during this waiting period, or additional time canoptionally be provided for clearing of the unbound modified red bloodcells from non-target tissue. The waiting period will be determinedclinically and may vary depending on the composition of the composition.

At the conclusion of this waiting period, a light source is used toexcite the bound photoactivatable molecule. The light source may providenon-coherent (non-laser) or coherent (laser) light. For example,non-coherent light sources include, but are not limited to, mercury orxenon arc lamps with optical filters, tungsten lamps, cold cathodefluorescent lamps, halogen lamps, light emitting diodes (LEDs), LEDarrays, incandescent sources, and other electroluminescent devices. Lampsources are used when fine definition of the illumination region is notrequired, or when a large region is to be illuminated. Focusednon-coherent light can be used to illuminate small regions, such as byusing lenses to focus the light or optical fibers to direct or deliverthe light. Laser sources are usually used to illuminate small,well-defined regions, because of their higher specific radiance and morereadily controlled beam properties. Coherent light sources include, butare not limited to, dye lasers, argon ion lasers, laser diodes, tunablelasers, Ti-sapphire lasers, Ruby lasers, Alexandrite lasers, Helium-Neonlasers, GaAlAs and InGaAs diode lasers, Nd-YLF lasers, Nd-glass lasers,Nd-YAG lasers and fiber lasers. For example, lasers are often used asexcitation sources in confocal equipment, and to create very high flux.Laser sources are limited in that they emit a restricted, often discreteset of wavelengths in contrast to lamps, which generally produce acontinuous spectrum that can be filtered to provide any desired bandwithin a certain range.

The area of illumination is determined by the location and dimension ofthe pathologic region to be detected, diagnosed or treated. The durationof illumination period will depend on whether detection or treatment isbeing performed, and can be determined empirically. A total orcumulative period of time anywhere from between about 1 min and 72 h canbe used. In an embodiment, the illumination period is between about 4min and 48 h. In another embodiment, the illumination period is betweenabout 30 min and 24 h.

The total fluence (i.e., power) or energy of the light used forirradiating is from about 10 Joules and about 25,000 Joules; in anembodiment, the total fluence is from about 100 Joules and about 20,000Joules or from about 500 Joules and about 10,000 Joules. Light of awavelength and fluence sufficient to produce the desired effect isselected, whether for detection by fluorescence or for therapeutictreatment to destroy or impair a target tissue or target cell. Lighthaving a wavelength corresponding at least in part with thecharacteristic light absorption wavelength of the photosensitizing agentis used for irradiating the target issue.

The power delivered by the light used is measured in watts, where 1 wattis equal to 1 joule/sec. Intensity is the power per area. Thus,intensity may be measured in watts/cm². Therefore, the intensity of thelight used for irradiating may be between about 5 mW/cm² to about 500mW/cm². Since the total fluence or amount of energy of the light inJoules is divided by the duration of total exposure time in seconds, thelonger the amount of time the target is exposed to the irradiation, thegreater the amount of total energy or fluence may be used withoutincreasing the amount of the intensity of the light used. The methodstypically employ an amount of total fluence of irradiation that issufficiently high to excite the photoactivatable molecule of thetarget-binding agent.

In an embodiment of using the modified red blood cells disclosed hereinfor photodynamic therapy, the modified red blood cells are injected intothe mammal, e.g., human, to be diagnosed or treated. The level ofinjection is usually between about 0.1 and about 0.5 μmol/kg of bodyweight. In the case of treatment, the area to be treated is exposed tolight at the desired wavelength and energy, e.g., from about 10 to 200J/cm² In the case of detection, fluorescence is determined upon exposureto light at a wavelength sufficient to cause the target-binding agent tofluoresce at a wavelength different than that used to illuminate theconjugate. The energy used in detection is sufficient to causefluorescence and is usually significantly lower than is required fortreatment.

The following sections will describe particular diseases or conditionsthat may be treated using the modified red blood cells.

A. Methods of Treating Cancer and Other Hyperproliferative Disorders

A neoplasm or tumor is an abnormal tissue growth resulting fromneoplastic cells, i.e., cells that proliferate more.rapidly anduncontrollably than normal cells. Usually partially or completelystructurally disorganized, neoplasms lack functional coordination withthe corresponding normal tissue. Neoplasms usually form a distincttissue mass that may be either benign (tumor) or malignant (cancer). Inaddition to structural disorganization, cancer cells usually regress tomore primitive or undifferentiated states (anaplasia), althoughmorphologically and biochemically, they may still exhibit many functionsof the corresponding wild-type cells. Carcinomas are cancers derivedfrom epithelia; sarcomas are derived from connective tissues. In somecases, cancers may not be associated with a tumor, but like the affectedtissue, is defuse, e.g., leukemias.

The modified red blood cells may be used to target neoplastic cells anddesignate those cells for damage or destruction. For example, thephotoactivatable molecule of the modified red blood cells may act uponthe neoplastic cells directly by bringing the cells in contact withsinglet oxygen. Alternatively, the modified red blood cells may comprisered blood cells loaded with one or more therapeutic agents, e.g., achemotherapeutic or antineoplastic agent, which is released from themodified red blood cell at the desired location. Consequently, theneoplastic cell is brought into close contact with a relatively highconcentration of the therapeutic agent.

As described above, the modified red blood cells may be loaded with oneor more chemotherapeutic agents for targeted delivery to a neoplasticcell. Examples of chemotherapeutic or antineoplastic agents include, butare not limited to, an alkylating agent; cisplatin; carboplatin;oxaliplatin; mechlorethamine; cyclophosphamide; chlorambucil;anti-metabolite compound; azathioprine; mercaptopurine; alkaloids;terpenoids; vinca alkaloid; vincristine; vinblastine; vinorelbine;vindesine; podophyllotoxin; taxanes; taxol; docetaxel; paclitaxel;topoisomerase inhibitors; camptothecins; irinotecan; topotecan;amsacrine; etoposide; etoposide phosphate; and teniposide;epipodophyllotoxins; antitumour antibiotics; dactinomycin; trastuzumab(Herceptin), cetuximab, and rituximab (Rituxan or Mabthera); Bevacizumab(Avastin); finasteride; tamoxifen; gonadotropin-releasing hormoneagonists (GnRH); and goserelin.

B. Methods of Treating a Pathogen Infections

1. Bacterial Infections

Bacteremia is the presence of bacteria in the blood. Bacteremia has manypossible causes, including dental procedures or even vigoroustoothbrushing; catheterization of an infected lower urinary tract;surgical treatment of an abscess or infected wound; and colonization ofindwelling devices, especially IV and intracardiac catheters, urethralcatheters, and ostomy devices and tubes. Gram-negative bacteremiasecondary to infection usually originates in the GU or GI tract, or theskin in patients with decubitus ulcers. Chronically ill andimmunocompromised patients have an increased risk of gram-negativebacteremia. They may also develop bacteremia with gram-positive cocci,anaerobes, and fungi. Staphylococcal bacteremia is common in injectiondrug users. Bacteroides bacteremia may develop in patients withinfections of the abdomen and the pelvis, particularly the femalegenital tract.

Metastatic infection of the meninges or serous cavities, such as thepericardium or larger joints, can result from transient or sustainedbacteremia. Metastatic abscesses may occur almost anywhere. Multipleabscess formation is especially common with staphylococcal bacteremia.Bacteremia may cause endocarditis, most commonly if the pathogen is anEnterococcus, Streptococcus, or Staphylococcus, and less commonly withgram-negative bacteremia and fungemia. Patients with valvular heartdisease, prosthetic heart valves, or other intravascular prostheses arepredisposed to endocarditis, which may occur after certain dentalprocedures. Staphylococci can cause gram-positive bacterialendocarditis, particularly in injection drug users, and may involve thetricuspid valve. The bacteria most likely to cause bacteremia includemembers of the Staphylococcus, Streptococcus, Pseudomonas, Haemophilus,and Esherichia coli (E. coli) genera.

Bacterial diseases or disorders that can be treated or prevented by theuse of the modified red blood cells include, but are not limited to,Mycobacteria, Rickettsia, Mycoplasma, Neisseria meningitides, Neisseriagonorrheoeae, Legionella, Vibrio cholerae, Streptococci, Staphylococcusaureus, Staphylococcus epidermidis, Pseudomonas aeruginosa,Corynobacteria diphtheriae, Clostridium spp., enterotoxigenic Eschericiacoli, and Bacillus anthracis. Other pathogens for which bacteremia hasbeen reported at some level include the following: Rickettsia,Bartonella henselae, Bartonella quintana, Coxiella burnetii, chlamydia,Mycobacterium leprae, Salmonella; shigella; Yersinia enterocolitica;Yersinia pseudotuberculosis; Legionella pneumophila; Mycobacteriumtuberculosis; Listeria monocytogenes; Mycoplasma spp.; Pseudomonasfluorescens; Vibrio cholerae; Haemophilus influenzae; Bacillusanthracis; Treponema pallidum; Leptospira; Borrelia; Corynebacteriumdiphtheriae; Francisella; Brucella melitensis; Campylobacter jejuni;Enterobacter; Proteus mirabilis; Proteus; and Klebsiella pneumoniae.

A red blood cell may be modified with a moiety that allows the cell totarget bacteria existing in the blood or at precise locations within thebody. A target recognition moiety may be, for example, an antibody,antibody fragment, single chain antibody, DNA and/or RNAoligonucleotide, leptin, peptide, peptide nucleic acid (PNA), protein,receptor, drug, ligand, enzyme, and/or substrate, that is capable ofspecifically binding a target molecule associated with a bacteria.

In an embodiment, a red blood cell may be modified with a targetingantibody that specifically recognizes and targets the modified red bloodcell to bacteria (See, e.g., U.S. Pat. No. 6,506,381 B1; U.S. PatentApplication 2004/0033232 A1; U.S. Patent Application 2006/0018912 A1).The targeting antibody directed against a specific marker on the surfaceof the target cell may be generated using standard procedures.Alternatively, the targeting antibody may be commercially available.

One or more red blood cells may be modified with a target recognitionmoiety that is a cellular receptor that recognizes and/or binds tobacteria. For example, CD14, which is normally associated withmonocyte/macrophages is known to bind lipopolysaccharide associated withgram negative bacteria as well as lipoteichoic acid associated with thegram positive bacteria Bacillus subtilis (See, e.g., Fan et al., Infect.Immun. 67:2964-2968 (1999)). Other examples of cellular receptorsinclude but are not limited to adenylate cyclase (Bordatella pertussis),Gal alpha 1-4Gal-containing isoreceptors (E. coli), glycoconjugatereceptors (enteric bacteria), Lewis(b) blood group antigen receptor(Heliobacter pylori), CR3 receptor, protein kinase receptor, galactoseN-acetylgalactosamine-inhibitable lectin receptor, and chemokinereceptor (Legionella), annexin I (Leishmania mexicana), ActA protein(Listeria monocytogenes), meningococcal virulence associated Opareceptors (Meningococcus), {acute over (α)}5β3 integrin (Mycobacteriumavium-M), heparin sulphate proteoglycan receptor, CD66 receptor,integrin receptor, membrane cofactor protein, CD46, GM1, GM2, GM3, andCD3 (Neisseria gonorrhoeae), KDEL receptor (Pseudomonas), epidermalgrowth factor receptor (Samonella typhiurium), β1 integrin (Shigella),and nonglycosylated J774 receptor (Streptococci) (See, e.g., U.S. PatentApplication 2004/0033584 A1).

A modified red blood cell may include an antibody or aptamer that bindsa specific bacterium of interest. As such, the antibody may bring thered blood cell into close proximity to the bacteria. The red blood cellmay be further modified with an additional component that has theability to breach the outer membrane/cell wall of the bacterium such as,for example, lysozymes, bacteriocidal permeability increasing peptides,and other pore forming antimicrobials (See, e.g., U.S. Pat. No.6,506,381 B1). For example, Zaitsev et al., (Blood 108:1895-1902 (2006))describe methods for modifying a red blood cell with a serine proteaseby linking the protease to an antibody to CR1, an abundant proteincomponent of the red blood cell membrane. In this instance, the serineprotease, tissue plasminogen activator (tPA), attached to the red bloodcells retained its enzymatic activity in vivo. As such, lysozyme whichhydrolyses 1,4-beta-linkages between N-acetylmuramic acid andN-acetyl-D-glucosamine residues in a peptidoglycan and betweenN-acetyl-D-glucosamine residues in chitodextrins of some bacteria may besimilarly attached to the surface of a modified red blood cell throughconjugation to a red blood cell binding antibody, for example.Alternatively, lysozyme may be expressed on the surface of a modifiedred blood cell as part of a membrane associated fusion protein, forexample. Fusion proteins containing lysozyme have been described (See,e.g., U.S. Pat. Nos. 5,993,809 and 7,045,677). In addition, fusionproteins have been described that include a secreted protein that isretained in association with the exterior of a cell by fusion to aprotein with a membrane anchor domain (See, e.g., U.S. PatentApplication 2006/0068388 A1).

Alternatively, a modified red blood cell may include an antibody oraptamer that binds a specific bacterium of interest. In addition, themodified red blood cell may include one or more additional antibodiesand/or aptamers to which is reversibly attached a therapeutic agent(See, e.g., U.S. Patent Application 2003/0215454 A1). In some instances,the red blood cell binds to its target and due to the concave nature ofthe red blood cell creates a small volume of space into which a subsetof the therapeutic agent may diffuse to establish an equilibrium. Astherapeutic agent is taken up by the target cell, more therapeutic agentis released from the modified red blood cell.

The modified red blood cell may be modified with an antibody and/oraptamer, for example, that specifically binds a target cell. Uponbinding to the modified red blood cell, the target cell is immobilizedand may be cleared in accordance with the body's red blood cell clearingmechanism through the phagocytic cells of the reticuloendothelialsystem.

In some instances, a therapeutic agent may be selectively released froma modified red blood cell using ultrasound energy. For example, redblood cells that have been sensitized with an electrical field are moresensitive to ultrasound-induced disruption of the cell membrane thannormal, untreated red blood cells (See, e.g., U.S. Patent Application2004/0071664). As such, modified red blood cells loaded with atherapeutic agent may be sensitized ex vivo with an electrical fieldprior to transfusion of the cells into an individual. The sensitizingelectrical field may be as strong as that used for electroporation of atherapeutic agent into a modified red blood cell. Alternatively, lowerelectrical field strengths may be used. In general, electrical fieldstrengths may range, for example, from about 0.1 kVolts/cm to about 10kVolts/cm (See, e.g., U.S. Patent Applications 2002/0151004 A1 and2004/0071664 A1). Ultrasound energy with a power density ranging from0.05 to 100 W cm⁻² and frequency ranging from 0.015 to 10.0 MHz over atime frame ranging from 10 milliseconds to 60 minutes may be used todisrupt the sensitized red blood cells and induce release of the loadedtherapeutic agent (See, e.g., U.S. Patent Application 2004/0071664). Thesensitization step may be combined with the loading step using aspecific device such as that described in U.S. Pat. No. 6,495,351 B2.

Examples of therapeutic agents (i.e., antibiotics) include, but are notlimited to, beta-lactam compounds (penicillin, methicillin, nafcillin,oxacillin, cloxacillin, dicloxacilin, ampicillin, ticarcillin,amoxicillin, carbenicillin, piperacillin); cephalosporins & cephamycins(cefadroxil, cefazolin, cephalexin, cephalothin, cephapirin, cephradine,cefaclor, cefamandole, cefonicid, cefuroxime, cefprozil, loracarbef,ceforanide, cefoxitin, cefmetazole, cefotetan, cefoperazone, cefotaxime,ceftazidine, ceftizoxine, ceftriaxone, cefixime, cefpodoxime, proxetil,cefdinir, cefditoren, pivoxil, ceftibuten, moxalactam,cefepime); otherbeta-lactam drugs (aztreonam, clavulanic acid, sulbactam, tazobactam,ertapenem, imipenem, meropenem); cell wall membrane active agents(vancomycin, teicoplanin, daptomycin, fosfomycin, bacitracin,cycloserine); tetracyclines (tetracycline, chlortetracycline,oxytetracycline, demeclocycline, methacycline, doxycycline, minocycline,tigecycline); macrolides (erythromycin, clarithromycin, azithromycin,telithromycin); clindamycin; choramphenicol; quinupristin-dalfopristin;linezolid; aminoglycosides (streptomycin, neomycin, kanamycin, amikacin,gentamicin, tobramycin, sisomicin, netilmicin); spectinomycin;sulfonamides (sulfacytine, sulfisoxazole, silfamethizole, sulfadiazine,sulfamethoxazole, sulfapyridine, sulfadoxine); trimethoprim;pyrimethamine; trimethoprim-sulfamethoxazole; fluoroquinolones(ciprofloxacin, gatifloxacin, gemifloxacin, levofloxacin, lomefloxacin,moxifloxacin, norfloxacin, ofloxacin); colistimethate sodium,methenamine hippurate, methenamine mandelate, metronidazole, mupirocin,nitrofurantoin, and polymyxin B. Examples of anti-mycobacteria drugsinclude, but are not limited to: isoniazid, rifampin, rifabutin,rifapentine, pyrazinamide, ethambutol, ethionamide, capreomycin,clofazimine, and dapsone.

2. Methods of Treating Fungal Infection

Fungemia (also known as candidemia, candedemia, and invasivecandidiasis) is the presence of fungi or yeasts in the blood. The mostcommonly known pathogen is Candida albicans, causing roughly 70% offungemias, followed by Candida glabrata with 10%, and Aspergillus with1%. However, the frequency of infection by T. glabrata, Candidatropicalis, C. krusei, and C. parapsilosis is increasing, especiallywhen significant use of fluconazole is common.

A red blood cell may be modified with a moiety that allows the cell totarget fungal cells in the subject's body. A target recognition moietymay be, for example, an antibody, antibody fragment, single chainantibody, DNA and/or RNA oligonucleotide, leptin, peptide, peptidenucleic acid (PNA), protein, receptor, drug, ligand, enzyme, and/orsubstrate, that is capable of specifically binding a target moleculeassociated with a fungal cell.

In an embodiment, a red blood cell may be modified with a targetingantibody that specifically recognizes and targets the modified red bloodcell to fungi. The targeting antibody directed against a specific markeron the surface of the target cell may be generated using standardprocedures. Alternatively, the targeting antibody may be commerciallyavailable.

In an embodiment, a modified red blood cell may be loaded with anantifungal agent that is released upon contact with the fungal cell.Examples of antifungal agents includes, but is not limited to:allylamines; terbinafine; antimetabolites; flucytosine; azoles;fluconazole; itraconazole; ketoconazole; ravuconazole; posaconazole;voriconazole; glucan synthesis inhibitors; caspofungin; micafungin;anidulafungin; polyenes; amphotericin B; amphotericin B Lipid Complex(ABLC); amphotericin B Colloidal Dispersion (ABCD); liposomalamphotericin B (L-AMB); liposomal nystatin; and griseofulvin.

3. Methods of Treating a Parasitic Infection

In an embodiment, the modified red blood may be administered to asubject for the treatment of a parasitic infection. The targetingcompositions may be directed to intestinal or blood-borne parasites,including protazoa. Typically, blood-borne parasites are transmittedthrough an arthropod vector. Most important arthropod for transmittingparasitic infections are mosquitoes. Mosquitoes carry malaria andfilarial nematodes. Biting flies transmit African trypanosomiasis,leishmaniasis and several kinds of filariasis. Examples of parasitesinclude, but are not limited to, trypanosomes; haemoprotozoa andparasites capable of causing malaria; enteric and systemic cestodesincluding taeniid cestodes; enteric coccidians; enteric flagellateprotozoa; filarial nematodes; gastrointestinal and systemic nematodesand hookworms.

A red blood cell may be modified with a moiety that allows the cell totarget the parasite or particular cells of the parasite. A targetrecognition moiety may be, for example, an antibody, antibody fragment,single chain antibody, DNA and/or RNA oligonucleotide, leptin, peptide,peptide nucleic acid (PNA), protein, receptor, drug, ligand, enzyme,and/or substrate, that is capable of binding a target moleculeassociated with the parasite.

In an embodiment, a red blood cell may be modified with a targetingantibody that recognizes and targets the red blood cell to the parasite.The targeting antibody directed against a marker on the surface of thetarget may be generated using standard procedures. Alternatively, thetargeting antibody may be commercially available.

In an embodiment, a modified red blood cell may be loaded with ananti-parasitic agent that is released upon contact with the parasite.Examples of anti-parasitic drugs include, but are not limited to:antiprotozoal agents; eflornithine; furazolidone; melarsoprol;metronidazole; ornidazole; paromomycin sulfate; pentamidine;pyrimethamine; tinidazole; antimalarial agents; quinine; chloroquine;amodiaquine; pyrimethamine; sulphadoxine; proguanil; mefloquine;halofantrine; primaquine; artemesinin and derivatives thereof;doxycycline; clindamycin; benznidazole; nifurtimox; antihelminthics;albendazole; diethylcarbamazine; mebendazole; niclosamide; ivermectin;suramin; thiabendazole; pyrantel pamoate; levamisole; piperazine family;praziquantel; triclabendazole; octadepsipeptides; and emodepside.

4. Methods of Treating a Viral Infection

In an embodiment, the modified red blood may be administered to asubject for the treatment of a viral infection. A red blood cell may bemodified with a moiety that allows the cell to target the virus or hostcells of the virus. A target recognition moiety may be, for example, anantibody, antibody fragment, single chain antibody, DNA and/or RNAoligonucleotide, leptin, peptide, peptide nucleic acid (PNA), protein,receptor, drug, ligand, enzyme, and/or substrate, that is capable ofspecifically binding a target molecule associated with the virus.

In an embodiment, a red blood cell may be modified with a targetingantibody that specifically recognizes and targets the red blood cell tothe virus. The targeting antibody directed against a specific marker onthe surface of the virus may be generated using standard procedures.Alternatively, the targeting antibody may be commercially available. Forexample, the target recognition moieties of the modified red blood cellsmay be directed to clinically important viruses, including but notlimited to adenovirus, coxsackievirus, hepatitis a virus, poliovirus,epstein-barr virus, herpes simplex, type 1, herpes simplex, type 2,human cytomegalovirus, human herpesvirus, type 8, varicella-zostervirus, hepatitis B virus, hepatitis C viruses, human immunodeficiencyvirus (HIV), influenza virus, measles virus, mumps virus, parainfluenzavirus, respiratory syncytial virus, papillomavirus, rabies virus, andRubella virus.

In an embodiment, a modified red blood cell may be loaded with anantiviral agent that is released upon contact with the virus. Examplesof antiviral agents include: thiosemicarbazones; metisazone; nucleosidesand nucleotides; acyclovir; idoxuridine; vidarabine; ribavirin;ganciclovir; famciclovir; valaciclovir; cidofovir; penciclovir;valganciclovir; brivudine; ribavirin, cyclic amines; rimantadine;tromantadine; phosphonic acid derivatives; foscarnet; fosfonet; proteaseinhibitors; saquinavir; indinavir; ritonavir; nelfinavir; amprenavir;lopinavir; fosamprenavir; atazanavir; tipranavir; nucleoside andnucleotide reverse transcriptase inhibitors; zidovudine; didanosine;zalcitabine; stavudine; lamivudine; abacavir; tenofovir disoproxil;adefovir dipivoxil; emtricitabine; entecavir; non-nucleoside reversetranscriptase inhibitors; nevirapine; delavirdine; efavirenz;neuraminidase inhibitors; zanamivir; oseltamivir; moroxydine; inosinepranobex; pleconaril; and enfuvirtide.

III. Diagnostic and Imaging Methods

In one aspect, the disclosure provides methods of using modified redblood cells to deliver an imaging agent to a cell or a tissue within asubject. In an embodiment, the modified red blood cells may beengineered to express or carry one or more molecular markers; whereinthe one or more molecular markers are configured to be activated byinteraction with one or more molecules to be detected. For example, anaptamer-based molecular beacon may be located in the cytoplasm of a redblood cell and detect changes in cellular signaling associated withinteraction of a modified red blood cell with a target.

In another embodiment, the red blood cells are loaded with an imagingagent that emits a detectable signal, such as light or otherelectromagnetic radiation. In another embodiment, the imaging agent is aradio-isotope, for example ³²P or ³⁵S or ⁹⁹Tc, or a molecule such as anucleic acid, polypeptide, or other molecule, conjugated with such aradio-isotope. In an embodiment, the imaging agent is opaque toradiation, such as X-ray radiation. For example, the agent may comprisea radiolabelled antibody which specifically binds to definedmolecule(s), tissue(s) or cell(s) in an organism.

In another embodiment, the imaging agent is a contrast dye. For example,an MRI contrast agent can comprise a paramagnetic contrast agent (suchas a gadolinium compound), a superparamagnetic contrast agent (such asiron oxide nanoparticles), a diamagnetic agent (such as barium sulfate),and combinations thereof. Metal ions preferred for MRI include thosewith atomic numbers 21-29, 39-47, or 57-83, and, more typically, aparamagnetic form of a metal ion with atomic numbers 21-29, 42, 44, or57-83. Particularly preferred paramagnetic metal ions are selected fromthe group consisting of Gd(III), Fe(III), Mn(II and III), Cr(III),Cu(II), Dy(III), Tb(III and IV), Ho(III), Er(III), Pr(III) and Eu(II andIII). Gd(III) is particularly useful. Note that as used herein, the term“Gd” is meant to convey the ionic form of the metal gadolinium; such anionic form can be written as GD(III), GD3+, etc. with no difference inionic form contemplated. A CT contrast agent can comprise iodine (ionicor non-ionic formulations), barium, barium sulfate, Gastrografin (adiatrizoate meglumine and diatrizoate sodium solution), and combinationsthereof. In another embodiment, a PET or SPECT contrast agent cancomprise a metal chelate. Following administration of the contrast dye,the subject can be imaged using X-ray, MRI, CT, or PET scanning.

The compositions and methods described herein are further illustrated bythe following examples, which should not be construed as limiting in anyway.

EXAMPLES Example 1 Treatment of a Hyperproliferative Disorder

In this example, the modified red blood cells are used to treat ahyperproliferative disorder (e.g., cancer). In one instance, themodified red blood cells are targeted to surface antigens on aneoplastic cell, known or suspected to be present in a subject's body.Upon excitation with light of the appropriate wavelength and power,singlet oxygen radicals are generated, resulting in damage ordestruction of the neoplastic cell. In another instance, the modifiedred blood cells are used to deliver a therapeutic agent to theneoplastic cell(s).

A photoactivatable molecule, such as a porphyrin, is conjugated, via anamide linkage, to a monoclonal antibody known to exhibit selectivebinding to an antigen expressed on the surface of a neoplastic cell. Theantibody is also conjugated to a quenching agent such as a Dabcyl(4-(4′-dimethylaminophenylazo)benzoyl) group, by reaction with acommercially available agent such as dabcyl chloride. Thistarget-binding agent is further modified by the addition of a suitablemetal ion to an aqueous solution of the composition. The metal binds tothe coordination pocket of the porphyrin ring-system and alsocoordinates the amine or azo group of the quenching group, ensuring thatthe quenching agent remains sufficiently close to the photoactivatablemolecule to allow energy transfer and thereby quench the generation ofsinglet oxygen.

Next, red blood cells are isolated from the subject in need oftreatment. In cases where it is desirable to deliver a therapeutic agentto the neoplastic cells, the cells are loaded with a chemotherapeuticagent, such as 5-fluorouracil, and biotinylated. The antibody is alsoconjugated with biotin and then linked to the biotinylated red bloodcells by a streptavidin bridge to form an assembled target-bindingagent. The assembled target-binding agent is mixed with a suitableexcipient for intravenous administration to the subject. Atherapeutically effective amount of this target-binding agent isadministered to the subject.

Binding of the antibody to its target then disrupts the coordinationbinding environment, releasing the quencher molecule from the metal andallowing the quencher molecule to move away from the photoactivatablemolecule, thereby activating the target-binding agent. After asufficient time for the target-binding agent to bind to the intendedtarget and clear from normal tissue, a light source of the appropriatewavelength is used to deliver a therapeutically useful amount of lightto an area that includes the lesion or region of hyperproliferativetissue. The light causes the excitation of the photoactivable moiety,resulting in the production of a singlet oxygen radical molecule. Thesinglet oxygen radical molecule may act directly on the neoplastic cell,thereby damaging or destroying the cell. Alternatively, the singletoxygen radical disrupts the cell membrane of the red blood cell, therebyreleasing the chemotherapeutic agent. The chemotherapeutic agent iscontacted with the neoplastic cell causing cell death.

The efficacy of treatment is assessed by reduction in the number ofneoplastic cells or absence of the neoplastic cells; reduction in thetumor size; inhibition (i.e., slow to some extent and preferably stop)of tumor metastasis; inhibition, to some extent, of tumor growth;increase in length of remission, and/or relief to some extent, one ormore of the symptoms associated with the specific cancer.

Example 2 Treatment of a Pathogen Infection

In this example, the modified red blood cells are used to treat apathogen infection (e.g, bacterial, fungal, viral or parasitic). In oneinstance, the modified red blood cells are targeted to surface antigensof the pathogen, where upon excitation with light of the appropriatewavelength and power, singlet oxygen radicals are generated, resultingin damage or destruction of the pathogen. In another instance, themodified red blood cells are used to deliver a therapeutic agent to thepathogen, known or suspected to have infected a subect.

A photoactivatable molecule, such as a porphyrin, is conjugated, via anamide linkage, to a monoclonal antibody known to exhibit selectivebinding to an antigen expressed on the surface of a pathogen, e.g., thebacterium Staphylococcus aureus. The antibody is also conjugated to aquenching agent such as a Dabcyl (4-(4′-dimethylaminophenylazo)benzoyl)group, by reaction with a commercially available agent such as dabcylchloride. This target-binding agent is further modified by the additionof a suitable metal ion to an aqueous solution of the composition. Themetal binds to the coordination pocket of the porphyrin ring-system andalso coordinates the amine or azo group of the quenching group, ensuringthat the quenching agent remains sufficiently close to thephotoactivatable molecule to allow energy transfer and thereby quenchthe generation of singlet oxygen.

Next, red blood cells are isolated from the subject in need oftreatment. In cases where it is desirable to use a therapeutic agent,the cells are loaded with the therapeutic agent (e.g., an antibiotic,antifungal, antiparasitic, or antiviral), such as ciprofloxacin, andbiotinylated. The antibody is also conjugated with biotin and thenlinked to the biotinylated red blood cells by a streptavidin bridge toform an assembled target-binding agent. The assembled target-bindingagent is mixed with a suitable excipient for intravenous administrationto the subject. A therapeutically effective amount of thistarget-binding agent is administered to the subject.

Binding of the antibody to its pathogen target then disrupts thecoordination binding environment, releasing the quencher molecule fromthe metal and allowing the quencher molecule to move away from thephotoactivatable molecule, thereby activating the target-binding agent.After a sufficient time for the target-binding agent to bind to theintended target and clear from normal tissue, a light source of theappropriate wavelength is used to deliver a therapeutically usefulamount to light to an area that includes the lesion. The light causesthe excitation of the photoactivable moiety, resulting in the productionof a singlet oxygen radical molecule. The singlet oxygen radicalmolecule may act on pathogen directly, thereby damaging or destroyingthe pathogen. Alternatively, the singlet oxygen radical disrupts thecell membrane of the red blood cell, which has been loaded with thetherapeutic agent, thereby releasing the therapeutic agent. Thetherapeutic agent is contacted with the pathogen causing damage, deathor inactivation of the pathogen.

The efficacy of treatment is assessed by reduction in the number ofpathogen cells or absence of the pathgen cells; or reduction one or moreof the symptoms associated with the infection.

Example 3 Imaging a Target Tissue of a Subject

In this example, the modified red blood cells are used to transport animaging agent, i.e. fluorescent molecule or radiocontrast dye, to aparticular tissue or cell-type. A photoactivatable molecule, such as aporphyrin, is conjugated, via an amide linkage, to a monoclonal antibodyknown to exhibit selective binding to an antigen expressed in aparticular tissue of the subject. The antibody is also conjugated to aquenching agent such as a Dabcyl (4-(4′-dimethylaminophenylazo)benzoyl)group, by reaction with a commercially available agent such as dabcylchloride. This target-binding agent is further modified by the additionof a suitable metal ion to an aqueous solution of the composition. Themetal binds to the coordination pocket of the porphyrin ring-system andalso coordinates the amine or azo group of the quenching group, ensuringthat the quenching agent remains sufficiently close to thephotoactivatable molecule to allow energy transfer and thereby quenchthe generation of singlet oxygen.

Next, red blood cells are isolated from the subject in need of imaging.The cells are loaded with the imaging agent and biotinylated. Theantibody is also conjugated with biotin and then linked to thebiotinylated red blood cells by a streptavidin bridge to form anassembled modified red blood cell. The assembled modified red blood cellis mixed with a suitable excipient for intravenous administration to thesubject. A therapeutically effective amount of this modified red bloodcell is administered to the subject.

Binding of the antibody to its bacterial target then disrupts thecoordination binding environment, releasing the quencher molecule fromthe metal and allowing the quencher molecule to move away from thephotoactivatable molecule, thereby activating the target-binding agent.After a sufficient time for the target-binding agent to bind to theintended target and clear from normal tissue, a light source of theappropriate wavelength is used to deliver a useful amount to light to anarea that includes the lesion. The light causes the excitation of thephotoactivable moiety, resulting in the production of a singlet oxygenradical molecule, which disrupts the cell membrane of the red bloodcell, thereby releasing the imaging agent, e.g., radiocontrast dye. Theimaging agent is detected using X-ray, CT, or other means.

Equivalents

The present disclosure is not to be limited in terms of the particularembodiments described in this application, which are intended as singleillustrations of individual aspects of. Many modifications andvariations can be made without departing from its spirit and scope, aswill be apparent to those skilled in the art. Functionally equivalentmethods and apparatuses within the scope of the disclosure, in additionto those enumerated herein, will be apparent to those skilled in the artfrom the foregoing descriptions. Such modifications and variations areintended to fall within the scope of the appended claims. The presentdisclosure is to be limited only by the terms of the appended claims,along with the full scope of equivalents to which such claims areentitled. It is to be understood that this disclosure is not limited toparticular methods, reagents, compounds compositions or biologicalsystems, which can, of course, vary. It is also to be understood thatthe terminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting.

For any and all purposes, particularly in terms of providing a writtendescription, all ranges disclosed herein also encompass any and allpossible subranges and combinations of subranges thereof. Any listedrange can be easily recognized as sufficiently describing and enablingthe same range being broken down into at least equal halves, thirds,quarters, fifths, tenths, etc. As a non-limiting example, each rangediscussed herein can be readily broken down into a lower third, middlethird and upper third, etc. All language such as “up to,” “at least,”“greater than,” “less than,” and the like include the number recited andrefer to ranges which can be subsequently broken down into subranges asdiscussed above. Finally, a range includes each individual member. Thus,for example, a group having 1-3 cells refers to groups having 1, 2, or 3cells. Similarly, a group having 1-5 cells refers to groups having 1, 2,3, 4, or 5 cells, and so forth.

The various aspects and embodiments disclosed herein are for purposes ofillustration and are not intended to be limiting, with the true scopeand spirit being indicated by the following claims.

The various aspects and embodiments disclosed herein are for purposes ofillustration and are not intended to be limiting, with the true scopeand spirit being indicated by the following claims.

All publications and patent applications cited in this specification areherein incorporated by reference to the extent not inconsistent with thedescription herein and for all purposes as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference for all purposes.

The herein described components (e.g., steps), devices, and objects andthe description accompanying them are used as examples for the sake ofconceptual clarity and that various configuration modifications usingthe disclosure provided herein are within the skill of those in the art.Consequently, as used herein, the specific exemplars set forth and theaccompanying description are intended to be representative of their moregeneral classes. In general, use of any specific exemplar herein is alsointended to be representative of its class, and the non-inclusion ofsuch specific components (e.g., steps), devices, and objects hereinshould not be taken as indicating that limitation is desired.

With respect to the use of substantially any plural or singular termsherein, those having skill in the art can translate from the plural tothe singular or from the singular to the plural as is appropriate to thecontext or application. The various singular/plural permutations are notexpressly set forth herein for sake of clarity.

The herein described subject matter sometimes illustrates differentcomponents contained within, or connected with, different othercomponents. It is to be understood that such depicted architectures aremerely exemplary, and that in fact many other architectures can beimplemented which achieve the same functionality. In a conceptual sense,any arrangement of components to achieve the same functionality iseffectively “associated” such that the desired functionality isachieved. Hence, any two components herein combined to achieve aparticular functionality can be seen as “associated with” each othersuch that the desired functionality is achieved, irrespective ofarchitectures or intermedial components. Likewise, any two components soassociated can also be viewed as being “operably connected,” or“operably coupled,” to each other to achieve the desired functionality,and any two components capable of being so associated can also be viewedas being “operably couplable,” to each other to achieve the desiredfunctionality. Specific examples of operably couplable include but arenot limited to physically mateable or physically interacting componentsor wirelessly interactable or wirelessly interacting components orlogically interacting or logically interactable components.

While particular aspects of the present subject matter described hereinhave been shown and described, changes and modifications may be madewithout departing from the subject matter described herein and itsbroader aspects and, therefore, the appended claims are to encompasswithin their scope all such changes and modifications as are within thetrue spirit and scope of the subject matter described herein.Furthermore, it is to be understood that the invention is defined by theappended claims. It will be understood that, in general, terms usedherein, and especially in the appended claims (e.g., bodies of theappended claims) are generally intended as “open” terms (e.g., the term“including” should be interpreted as “including but not limited to,” theterm “having” should be interpreted as “having at least,” the term“includes” should be interpreted as “includes but is not limited to,”etc.). It will be further understood that if a specific number of anintroduced claim recitation is intended, such an intent will beexplicitly recited in the claim, and in the absence of such recitationno such intent is present. For example, as an aid to understanding, thefollowing appended claims may contain usage of the introductory phrases“at least one” and “one or more” to introduce claim recitations.However, the use of such phrases should not be construed to imply thatthe introduction of a claim recitation by the indefinite articles “a” or“an” limits any particular claim containing such introduced claimrecitation to inventions containing only one such recitation, even whenthe same claim includes the introductory phrases “one or more” or “atleast one” and indefinite articles such as “a” or “an”; the same holdstrue for the use of definite articles used to introduce claimrecitations. In addition, even if a specific number of an introducedclaim recitation is explicitly recited, such recitation should typicallybe interpreted to mean at least the recited number (e.g., the barerecitation of “two recitations,” without other modifiers, typicallymeans at least two recitations, or two or more recitations).Furthermore, in those instances where a convention analogous to “atleast one of A, B, and C, etc.” is used, in general such a constructionis intended in the sense one having skill in the art would understandthe convention (e.g., “a system having at least one of A, B, and C”would include but not be limited to systems that have A alone, B alone,C alone, A and B together, A and C together, B and C together, or A, B,and C together, etc.). In those instances where a convention analogousto “at least one of A, B, or C, etc.” is used, in general such aconstruction is intended in the sense one having skill in the art wouldunderstand the convention (e.g., “a system having at least one of A, B,or C” would include but not be limited to systems that have A alone, Balone, C alone, A and B together, A and C together, B and C together, orA, B, and C together, etc.). Virtually any disjunctive word and/orphrase presenting two or more alternative terms, whether in thedescription, claims, or drawings, should be understood to contemplatethe possibilities of including one of the terms, either of the terms, orboth terms. For example, the phrase “A or B” will be understood toinclude the possibilities of “A” or “B” or “A and B.”

1. A method for detecting a target in a biological sample, comprising:adding to the biological sample a red blood cell that binds to thetarget, the red blood cell including one or more fusion molecules, andat least one target-binding agent including a target recognition moiety,wherein the target recognition moiety is configured to recognize one ormore target cells, and wherein the one or more fusion molecules areconfigured to promote fusion of the red blood cell with the one or moretarget cells; and detecting the modified red blood cell.
 2. The methodof claim 1, wherein the red blood cell expresses one or more of the atleast one target-binding agent and the one or more fusion molecules. 3.The method of claim 1, wherein the at least one target-binding agent andthe one or more fusion molecules are associated with the cell surface ofthe red blood cell.
 4. The method of claim 1, wherein the one or morefusion molecules includes at least one of antigen; ligand; receptor;polyamide; peptide; carbohydrate; oligosaccharide; polysaccharide; lowdensity lipoprotein (LDL); an apoprotein of LDL; steroid; steroidderivative; hormone; hormone-mimic; lectin; drug; antibiotic; aptamer;DNA; RNA; lipid; an antibody; or an antibody-related polypeptide.
 5. Themethod of claim 1, wherein the one or more fusion molecules is asyncytin-1 protein.
 6. The method of claim 1, wherein the targetrecognition moiety includes at least one of antigen; ligand; receptor;polyamide; peptide; carbohydrate; oligosaccharide; polysaccharide; lowdensity lipoprotein (LDL); an apoprotein of LDL; steroid; steroidderivative; hormone; hormone-mimic; lectin; drug; antibiotic; aptamer;DNA; RNA; lipid; an antibody; or an antibody-related polypeptide.
 7. Themethod of claim 1, further comprising one or more activatable molecularmarkers, wherein the one or more activatable molecular markers isconfigured to be activated by an interaction of the modified red bloodcell with the one or more target cells to produce a detectable response.8. The method of claim 7, wherein the one or more activatable molecularmarkers is a photoactivatable molecular marker and the detectableresponse is the production of reactive singlet oxygen molecules.
 9. Themethod of claim 7, wherein the modified red blood cell is geneticallyengineered to express the one or more activatable molecular markers. 10.The method of claim 9, wherein the one or more activatable molecularmarkers is an aptamer based molecular beacon.
 11. The method of claim 1,wherein the at least one target-binding agent further comprises aphotoactivatable molecule and a quencher molecule coupled to the targetrecognition moiety, wherein the at least one target-binding agent isconfigured to emit at least one singlet oxygen radical molecule uponexposure to light of a suitable wavelength when the at least onetarget-binding agent is bound to the one or more target cells.
 12. Themethod of claim 11, wherein the photoactivatable molecule and thequencher molecule include a linker.
 13. The method of claim 12, whereinthe linker includes at least one of an oligonucleotide or a sulfonicacid.
 14. The method of claim 11, wherein the photoactivatable moleculeincludes at least one of a porphyrin; chlorin; bacteriochlorin;isbacteriochlorin; phthalocyanine; napthalocyanine; porphycene;porphycyanine; tetra-macrocyclic compound; poly-macrocyclic compound;pyropheo-phorbide; pentaphyrin; sapphyrin; texaphyrin; metal complexe;tetrahydrochlorin; phonoxazine dye; phenothiazine; chaloorganapyryliumdye; rhodamine; fluorescene; azoporphyrin; benzochlorin; purpurin;chlorophyll; verdin; triarylmethane; angelicin; chalcogenapyrillium dye;chlorin; chlorophyll; coumarin; cyanine; ceratin daunomycin;daunomycinone; 5-iminodauno-mycin; doxycycline; furosemide; gilvocarcinM; gilvocarcin V; hydroxy-chloroquine sulfate; lumidoxycycline;mefloquine hydrochloride; mequitazine; merbromin (mercurochrome);primaquine diphosphate; quinacrine dihydrochloride; quinine sulfate;tetracycline hydrochloride; flavin; alloxazine; flavin mononucleotide;3-hydroxyflavone; limichrome; limiflavin; 6-methylalloxazine;7-methylalloxazine; 8-methylalloxazine; 9-methylalloxazine; 1-methyllimichrome; methyl-2-methoxybenzoate; 5-nitrosalicyclic acid;proflavine; and riboflavin; metallo-porphyrin; metallophthalocyanine;methylene blue derivative; naphthalmide; naphthalocyanine; pheophorbide;pheophytin; photosensitizer dimer and conjugate; phthalocyanine;porphycene; quinone; retinoid; rhodamine; thiophene; verdin; vitamin; orxanthene dye.
 15. The method of claim 11, wherein the quencher moleculeincludes at least one of: 4-(4′-dimethylamino-phenylazo)benzoic acid(DABCYL); dabcyl succinimidyl ester;4-(4′-dimethylamino-phenylazo)sulfonic (DABSYL); dabsyl succinimidylester; tetramethyl-rhodamaine (TAMRA);4-[(4-nitrophenyl)diazinyl]-phenylamine and4-[4-nitrophenyl)diazinyl]-naphthylamine; dabcylnitro-thiazole;6-(N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl]amino) hexanoic acid;6-carboxy-X-rhodamine (ROX);QSY-7;2-[4-(4-nitrophenylazo)-N-ethylphenyl-amino]ethanol (Disperse Red 1);2-[4-(2-chloro-4-nitrophenyl-azo)-N-ethylphenylamino]-ethanol (DisperseRed 13); tetrarhodamine isothiocyanate (TRITC); allophycocyanin;β-carotene; diarylrhodamine derivatives, QSY 7, QSY 9, QSY 21 dyes; QSY35 acetic acid succinimidyl ester; QSY 35 iodoacetamide; aliphaticmethylamine; napthalate; Reactive Red 4; or Malachite Green.
 16. Themethod of claim 1, wherein the red blood cell includes a reticulocyte; ared blood cell; or a fusion between a red blood cell between a red bloodcell autologous to a subject and one or more allogeneic erythrocytes,liposomes or artificial vesicles.
 17. The method of claim 1, wherein thered blood cell is genetically engineered to express one or moreprotein-based pharmaceutical molecules or one or more RNA-basedpharmaceutical molecules.
 18. The method of claim 1, wherein the redblood cell is loaded with one or more pharmaceutical or imagingmolecules, or one or more viruses.
 19. The method of claim 18, whereinthe one or more pharmaceutical molecules includes at least one of anantibiotic, an antiviral agent, an antifungal agent, an anti-parasiticagent, an antibody, an antibody-related polypeptide, a antineoplasticagent; a protein-based pharmaceutical; or an RNA or DNA-basedpharmaceutical.
 20. The method of claim 19, wherein the antineoplasticagent includes at least one of an alkylating agent; cisplatin;carboplatin; oxaliplatin; mechlorethamine; cyclophosphamide;chlorambucil; anti-metabolite compound; azathioprine; mercaptopurine;alkaloid; terpenoid; vinca alkaloid; vincristine; vinblastine;vinorelbine; vindesine; podophyllotoxin; taxane; docetaxel; paclitaxel;topoisomerase inhibitor; camptothecin; irinotecan; topotecan; amsacrine;etoposide; etoposide phosphate; teniposide; epipodophyllotoxins;antitumour antibiotic; dactinomycin; trastuzumab, cetuximab, rituximab;bevacizumab; finasteride; tamoxifen; gonadotropin-releasing hormoneagonist (GnRH); or goserelin.
 21. The method of claim 19, wherein theantibiotic includes at least one of aminoglycoside; amikacin;gentamicin; kanamycin; neomycin; netilmicin; steptomycin; tobramycin;ansamycin; geldanamycin; herbimycin; carbacephem; loracarbef;carbacepenem; ertapenem; doripenem; imipenem/cilastatin; meropenem;cephalosporin; cefadroxil; cefazolin; cefalotin or cefalothin;cefalexin; cefaclor; cefamandole; cefoxitin; cefprozil; cefuroxime;cefixime; cefdinir; cefditoren; cefoperazone; cefotaxime; cefpodoxime;ceftazidime; ceftibuten; ceftizoxime; ceftriaxone; cefepime;ceftobiprole; glycopeptide; teicoplanin; vancomycin; macrolide;azithromycin; clarithromycin; dirithromycin; erythromicin;roxithromycin; troleandomycin; telithromycin; spectinomycin; monobactam;aztreonam; penicillin; amoxicillin; ampicillin; azlocillin;carbenicillin; cloxacillin; dicloxacillin; flucloxacillin; mezlocillin;meticillin; nafcillin; oxacillin; penicillin, piperacillin, ticarcillin;bacitracin; colistin; polymyxin B; quinolones; ciprofloxacin; enoxacin;gatifloxacin; levofloxacin; lomefloxacin; moxifloxacin; norfloxacin;ofloxacin; trovafloxacin; sulfonamide; mafenide; prontosil (archaic);sulfacetamide; sulfamethizole; sufanilimide (archaic); sulfasalazine;sulfisoxazole; trimethoprim; trimethoprim-sulfamethoxazole(co-trimoxazole) (TMP-SMX); tetracycline; demeclocycline; doxycycline;minocycline; oxytetracycline; tetracycline; arsphenamine;chloramphenicol; clindamycin; lincomycin; ethambutol; fosfomycin;fusidic acid; furazolidone; isoniazid; linezolid; metronidazole;mupirocin; nitrofuantoin; platensimycin; purazinamide;quinupristin/dalfopristin; rifampin or rifampicin; or tinidazole. 22.The method of claim 19, wherein the antiviral agent includes at leastone of thiosemicarbazone; metisazone; nucleoside, nucleotide; acyclovir;idoxuridine; vidarabine; ribavirin; ganciclovir; famciclovir;valaciclovir; cidofovir; penciclovir; valganciclovir; brivudine;ribavirin, cyclic amines; rimantadine; tromantadine; phosphonic acidderivatives; foscarnet; fosfonet; protease inhibitor; saquinavir;indinavir; ritonavir; nelfinavir; amprenavir; lopinavir; fosamprenavir;atazanavir; tipranavir; nucleoside or nucleotide reverse transcriptaseinhibitor; zidovudine; didanosine; zalcitabine; stavudine; lamivudine;abacavir; tenofovir disoproxil; adefovir dipivoxil; emtricitabine;entecavir; non-nucleoside reverse transcriptase inhibitor; nevirapine;delavirdine; efavirenz; neuraminidase inhibitors; zanamivir;oseltamivir; moroxydine; inosine pranobex; pleconaril; or enfuvirtide.23. The method of claim 19, wherein the anti-fungal agent includes atleast one of allylamine; terbinafine; antimetabolite; flucytosine;azole; fluconazole; itraconazole; ketoconazole; ravuconazole;posaconazole; voriconazole; glucan synthesis inhibitor; caspofungin;micafungin; anidulafungin; polyene; amphotericin B; amphotericin B LipidComplex (ABLC); amphotericin B Colloidal Dispersion (ABCD); liposomalamphotericin B (L-AMB); liposomal nystatin; or griseofulvin.
 24. Themethod of claim 19, wherein the anti-parasitic agent includes at leastone of antiprotozoal agent; eflornithine; furazolidone; melarsoprol;metronidazole; ornidazole; paromomycin sulfate; pentamidine;pyrimethamine; tinidazole; antimalarial agent; quinine; chloroquine;amodiaquine; pyrimethamine; sulphadoxine; proguanil; mefloquine;halofantrine; primaquine; artemesinin and derivatives thereof;doxycycline; clindamycin; benznidazole; nifurtimox; antihelminthics;albendazole; diethylcarbamazine; mebendazole; niclosamide; ivermectin;suramin; thiabendazole; pyrantel pamoate; levamisole; piperazine family;praziquantel; triclabendazole; octadepsipeptide; and emodepside.